(M0930-02-10) Debulking of SARS-CoV-2 and Influenza Virus Strains by Plant Viral Trap Proteins in Chewing Gums to Decrease Infection and Transmission

Purpose: SARS-CoV-2 an airborne pathogen is the causative agent for the on-going COVID-19 pandemic that replicates primarily in the salivary glands and mucosal surface linings of the oral cavity and oropharynx.¹ Although both nasal and oral passages are culpable, the latter with 3-5 higher magnitudes of viral load and saliva as a potential disseminator of virions remains a major source of viral transmission. This is further bolstered by the fact that four spoken words can release more virions than 1 hour of mask less breathing.^2,3 With global inequities in healthcare causing significant parts of the population not yet vaccinated, a novel drug delivery platform - protein drug infused chewing gums is well suited for neutralizing pathogens in the oral cavity and throat surfaces thus limiting viral transmission. We evaluate two plant based viral trap proteins namely CTB-ACE2 in lettuce and FRIL protein from Lablab Purpureus bean with distinct debulking mechanisms. While the CTB-ACE2 binds directly to the spike protein⁴ and blocks different SARS-CoV-2 variants, FRIL protein - a plant lectin entraps SARS-CoV-2 and influenza strains that express complex type N glycans on their outer envelopes.⁵ The debulking potential of both plant-based viral trap protein chewing gums are tested with different strains of coronaviruses (SARS-CoV-2, OC43), and influenza virus (H1N1, H3N2).

Methods: The debulking potential of both viral trap proteins were analyzed by the microbubbling SARS-CoV-2 antigen assay that works on the principle of nucleocapsid (N) antigen detection (Fig 1A, B). Patient nasopharyngeal (NP) swab samples with both Delta and Omicron variants of the SARS-CoV-2 were treated with varying doses of CTB-ACE2 gum (0.46, and 0.92 µg in 25 and 50 mg gum respectively) (Fig 1C) and FRIL protein (20, 40, 100 µg in 5, 10 and 25 mg bean powder respectively) (Fig 1D). The anti-viral effect of FRIL protein in its purified and crude form (bean powder) against various influenza viral strains – H1N1 (A/California/7/2009-X181), H3N2 (A/Singapore/INFMH-16/0019/2016), Coronavirus-HCoV-OC43 was tested using plaque reduction assay. The same technique was used to evaluate the viral inhibitory effect of FRIL chewing gum as compared to the placebo gum tablets. The viral aggregation mechanism of FRIL at 10 and 150 µg/mL was further evaluated using the negative-staining Electron Microscopy using H1N1 influenza strain.

Results: Both plant-based viral trap proteins – CTB-ACE2 and FRIL protein with distinct mechanisms were effective in debulking SARS-CoV-2 in patient NP swab samples at a minimum dose of 0.46 µg and 20 µg respectively. CTB-ACE2 gum (368.8 µg CTB-ACE2 /2 g tablet) at varying doses were effective against both delta and omicron variants of SARS-CoV-2, with significant reduction in the microbubble count (*** p-value < 0.0001) (Fig 1E, F). Omicron variant was effectively neutralized at varying doses of FRIL protein (4 mg FRIL /1 g bean powder) with significant reduction in the microbubble count (*** p-value < 0.0001) (Fig 1G). A midpoint inhibition of H1N1, H3N2, and OC43 with 95, 96 and 76 ng of purified FRIL protein was achieved. With lablab bean powder the midpoint inhibition was almost similar with 100 and 84 ng FRIL protein in 25 and 21 µg of bean powder respectively (Fig 2A-F). While we did not observe an inhibitory effect with the placebo gum, a midpoint inhibition with 555 mg of FRIL gum powder was noted (Fig 2G, H). The clustering of virions in large aggregates as seen in Fig 3 at 10-150 µg/mL of FRIL protein is due to the binding of carbohydrate-binding domain (CBD) to the complex-type N glycans on each of the four monomers thus connecting multiple virus particles.⁶

Conclusion: In summary, CTB-ACE2 is effective against a variety of SARS-CoV-2 strains and FRIL – a plant lectin is additionally effective against influenza strains. Lablab bean is commonly consumed globally⁷ and is generally regarded as safe (GRAS) by the FDA making it easier to advance to the clinic. Chewing gum delivery approach has been used for small molecules like aspirin, nicotine or for oral health purposes.^8,9,10 However, dispensing therapeutic proteins in gum base has not been successful owing to degradation of target proteins at high temperatures during gum manufacturing processes. Proteins encapsulated in plant cell walls, are resistant to high temperatures during rigorous gum production techniques, and remain stable during long-term storage at ambient temperature.¹¹ With the FDA approval of Emergency Use Authorization IND for CTB-ACE2 gum paving the way for Phase I/II Clinical trials, this revolutionary approach of delivering affordable plant engineered protein drugs is an invaluable tool to limit transmission of not only SARS-CoV-2 and influenza, but also other orally transmitted viruses.

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Acknowledgments: Research in the Daniell laboratory is supported by funding from NIH grant R01 HL 107904, R01 HL 109442, R01 HL 133191, Commonwealth of Pennsylvania, Department of Community and Economic Development grant and IDEA award. Authors thank Dr. Ronald G. Collman, Graham-Wooten. Jevon for providing NP patient samples, Wan Zhou and Ping Wang for assistance with microbubbling antigen assay, and Sudheer K. Molugu for assistance in Electron Microscopy.

Fig 1. Neutralization of SARS-CoV-2 Delta and Omicron variants in NP swab samples with CTB-ACE2 and FRIL viral trap proteins (A) Topical oral delivery of CTB-ACE2 (368.8 µg/2 g tablet) and FRIL plant viral trap protein (4 mg/1 g bean powder) infused chewing gums (B) Schematic of microbubble SARS-CoV-2 Antigen Assay (C) SARS-Cov-2 Omicron and Delta variants in patients NP samples treated with CTB-ACE2 gum (0.46 or 0.92 μg CTB-ACE2 protein in 25 and 50 mg of gum respectively) (D) SARS-Cov-2 Omicron variants in patients NP samples treated with FRIL bean powder (20,40,100 μg FRIL protein in 5,10 and 25 mg of bean powder respectively (E,F) CTB-ACE2 showed a significant reduction in the microbubble count in both variants (***p-value < 0.0001) (G) FRIL protein showed a significant reduction in the microbubble count (p < 0.0001) compared to untreated samples. Data were analyzed by One-way ANOVA test.



Fig 2. Dose dependent inhibition of plaque formation of H1N1 (A/California/7/2009-X181), H3N2 (A/Singapore/INFMH-16/0019/2016), and HCoV-OC43 virus with purified FRIL protein, lablab bean powder, Placebo and FRIL gum tablet (2 g tablet). (A-F) For purified FRIL protein, we see a midpoint inhibition with 95, 96 and 76 ng against H1N1, H3N2 and OC43 respectively. For lablab bean powder (4 mg FRIL protein/ 1g bean powder), we see a midpoint inhibition with 25 µg(100 ng FRIL protein), 21 µg(84 ng FRIL protein) and 13 µg(52 ng FRIL protein) against H1N1, H3N2 and OC43 respectively. Similar concentrations of purified FRIL protein and solubilized FRIL protein from lablab bean powder exerts similar midpoint inhibitory effect. This shows that the inhibitory effect of FRIL protein is independent of purification. The data represents Mean±SD of plaque numbers from two independent experiments in duplicate. (G,H) While we see a midpoint inhibition with 555 mg FRIL gum powder, the inhibitory effect of placebo gum is not significant at low concentrations.



Fig 3. FRIL entrapped Influenza Virus by Aggregation Negative-stain EM images of H1N1 (A/California/7/2009-X181) cell culture virions alone (left panels) and aggregated virus particles with purified FRIL after incubating with 10 µg/mL and 150 µg/mL FRIL (right panels). Data are representative of 2 independent experiments.