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Author(s): Henry Daniell, Yuwei Guo, Rahul Singh, Uddhab Karki, Rachel J. Kulchar, Geetanjali 
Wakade, Juha-Matti Pihlava, Hamid Khazaei, Gary H. Cohen 
Title: Debulking influenza and herpes simplex virus strains by a wide-spectrum anti-viral 
protein formulated in clinical grade chewing gum 
Year: 2025 
Version: Published version 
Copyright: The Author(s) 2025 
Rights: CC BY-NC-ND 4.0 
Rights url: https://creativecommons.org/licenses/by-nc-nd/4.0/  
 
Please cite the original version: 
Henry Daniell, Yuwei Guo, Rahul Singh, Uddhab Karki, Rachel J. Kulchar, Geetanjali Wakade, Juha-Matti 
Pihlava, Hamid Khazaei, Gary H. Cohen, Debulking influenza and herpes simplex virus strains by a 
wide-spectrum anti-viral protein formulated in clinical grade chewing gum, Molecular Therapy, 
Volume 33, Issue 1, 2025, Pages 184-200, ISSN 1525-0016, 
https://doi.org/10.1016/j.ymthe.2024.12.008.  
Original ArticleDebulking influenza and herpes simplex virus
strains by a wide-spectrum anti-viral protein
formulated in clinical grade chewing gum
Henry Daniell,1,4 Yuwei Guo,1,4 Rahul Singh,1 Uddhab Karki,1 Rachel J. Kulchar,1 Geetanjali Wakade,1
Juha-Matti Pihlava,2 Hamid Khazaei,2,3 and Gary H. Cohen1
1Department of Basic & Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; 2Natural Resources Institute Finland
(Luke), Helsinki, Finland; 3Department of Agricultural Sciences, University of Helsinki, Helsinki, FinlandLack of Herpes Simplex Virus (HSV) vaccine, low vaccination
rates of Influenza viruses, waning immunity and viral trans-
mission 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 15min of chew-
ing the bean gum. In plaque reduction assays, >95% neutraliza-
tion 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 vi-
rus 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 mini-
mize virus infection/transmission.Received 23 May 2024; accepted 6 December 2024;
https://doi.org/10.1016/j.ymthe.2024.12.008.
4These authors contributed equally
Correspondence: Dr. Henry Daniell, W. D. Miller Professor & Director of
Translational Research, Vice Chair, Department of Basic and Translational
Sciences, School of Dental Medicine, University of Pennsylvania, 240 South 40th
Street, 547 Levy Building, Philadelphia, PA 19104-6030, USA.
E-mail: hdaniell@upenn.eduINTRODUCTION
In today’s tightly connected world, infectious disease is becoming a
more severe and frequent threat due to several factors, including
climate changes, population density, increased international mobility,
and rapid urbanization in low-income countries with poor adoption
of public health measures.1,2 Of recent concern is the coronavirus
pandemic, in which severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2) led to 
7 million documented deaths worldwide.3
In addition, the past twenty years have witnessed several high-impact
infectious disease outbreaks (H1N1, SARS, Ebola, Zika virus) with
enormous global health burden and economic loss. In addition,
regional outbreaks with viruses like Influenza A H7N9 and H5N1,
Lassa fever virus, and MERS coronavirus have caused drastic local
losses.4 Improvement in infectious disease management measures is184 Molecular Therapy Vol. 33 No 1 January 2025 ª 2024 The Author(
Published by Elsevier Inc. on behalf of The American Society of Gen
This is an open access article under the CC BY-NC-ND license (httpmuch needed to prepare for the growing threat of infectious disease,
considering the absence of much-needed vaccines against viruses like
HSV and low vaccination rates for viruses like Influenza virus and
Ebola.5 The key to an effective disease control measure is reducing
viral loads in routes of transmission or at portals of viral entry
and exit.
Ample evidence suggested that intraoral viral load is correlated with
risk of disease transmission, with oral transmission 3-5 orders higher
than nasal transmission.6–9 Furthermore, the continuous infection of
COVID-19, irrespective of receiving booster vaccines and the recent
polio outbreaks in Israel and New York, highlights the need to address
their routes of transmission.10,11 Coronavirus, Influenza, HPV,
HSV1, EBV, and KSHV viruses are transmitted orally, and their life
cycle in the oral epithelium is well characterized.12–15 New methods
targeting intraoral viral load reduction are especially needed for epi-
demics where vaccines and therapeutics are not available. Effective
transmission prevention strategy becomes very important in the sce-
nario where almost 1 in 3 men are infected with at least one type of
genital HPV and 1 in 5 men are infected with one or more types of
high-risk HPV.16 Moreover, the frequent shedding of viruses from in-
fected individuals enhances the severity and complexity of the prob-
lem.17 For example, HSV-1 causes frequent shedding after the first
genital HSV-1 episode to up to one year.
Challenges remain for infection control regimens targeting viral load
reduction in the oral cavity, with the major limitation being that most
biologics can only be administered with injectable formulations.
Therefore, Daniell lab has developed a new approach utilizing chew-
ing gums through the encapsulation of protein drugs in plant cells,s).
e and Cell Therapy.
://creativecommons.org/licenses/by-nc-nd/4.0/).
Figure 1. Graphic Abstract: Engineering and evaluation of anti-viral bean gum
www.moleculartherapy.orgallowing for their oral topical delivery. Plant cell bioencapsulation
eliminates cold storage, transportation, purification challenges and
significantly reduces the cost of manufacturing biologics.18–24 The
chewing gum formulation allows for effective and consistent release
of the drug content at sites of viral infection. CTB-ACE2 chewing
gum was able to markedly debulk SARS-CoV-2 (>95%) in COVID-
19 patient saliva or swab samples.25,26 In addition, such efficacy was
consistent with various variants, including the most major beta, delta,
and omicron variants. These results supported an Investigational
New Drug approval from the U.S. Food and Drug Administration
for a Phase I/II clinical trial (IND 154897, NCT05433181, IRB
851459), which is aimed to evaluate the viral load reduction upon
chewing the gum amongst SARS-CoV-2 patients. This approach is
especially suitable for orally transmitted viruses, as it could reduce
viral load in saliva or the throat area, which is the main site of viral
transmission.
SARS-CoV-2, Influenza virus, HPV and HSV-1 are amongst the most
prevalent orally transmitted viruses. Seasonable Influenza epidemics
occur annually and incur substantial disease burden worldwide.14,27
It is estimated that more 32 million cases, 5–7 million hospitalization,
and 300,000 infection-related respiratory deaths occur worldwide
each year with over $11.2 billion loss annually in the United States
alone.5,27 Continuous efforts invested in vaccine development unfor-
tunately had limited returns, as from 2004 to 2020, vaccine effective-
ness among Influenza outpatients in the United States ranged from
10% to 60%.28 Vaccination efficacy is reduced by variation in anti-
genic match to circulating virus strains, low vaccination coverage
and disparities, and waning immunity among the elderly.29 On theother hand, HSV-1 affects over two-thirds of the world population,
with over 500,000 oral herpes cases each year in the United States
alone.30 HSV could induce encephalitis, accounting for 95% of en-
cephalitis cases, and is the leading cause of infectious blindness in
Western countries.31 Yet, no vaccines are currently approved for
HSV-1 nor HSV-2. In addition, methods beyond vaccination are
needed to control the transmission of these highly prevalent viruses.
Compared to other anti-viral proteins such as monocot lectin and
horcolin, Flt3 Receptor Interacting Lectin (FRIL), is significant in
its anti-viral properties against a wide range of pathogens.32 The car-
bohydrate-binding domain on each of four monomers of FRIL has
been documented to have preferential binding affinity to complex-
type N-glycans and oligosaccharides.33 In addition, the specificity of
FRIL differs from that ofMan/Glc binding lectins in that the mutation
inM3M6M trisaccharide allows FRIL subunits to cross-link and form
higher-order structures.32,34 Complex-type N-glycosylation is preva-
lent on enveloped viruses and preserved among variants as they
commonly serve as the mediator for viral entry, such as the spike pro-
tein of coronavirus and the hemagglutinin of Influenza virus.35–38
Therefore, the carbohydrate-binding domains on FRIL could bind
to the complex-type N-glycans on the viral envelope and thus entrap
virus in large aggregates of crosslinked FRIL and bound virion parti-
cles (Figure 1). FRIL has been documented to neutralize Influenza and
coronavirus as observed through plaque reduction and microbubble
assays.24 Furthermore, HSV-1 is proposed to enter epithelial cells
through four viral glycoproteins named gB, gD, gH, and gL.36,37
Several complex-type N-linked glycans present on gB are necessary
for optimal cell-cell fusion and entry.37,38Molecular Therapy Vol. 33 No 1 January 2025 185
Molecular TherapyUtilizing the delivery platform behind the success of the CTB-ACE2
gum, in this study we developed clinical-grade anti-viral chewing gum
using lablab bean powder and characterized drug product stability,
bioburden, moisture content, and antiviral efficacy to neutralize
Influenza (H1N1, H3N2) and HSV (HSV-1, HSV-2) viruses using
plaque reduction and ELISA assay (Figure 1). Together, the bean
powder chewing gum is prepared to comply with the FDA specifica-
tions for drug products and can be used to assess its potency to debulk
aforementioned orally transmitted viruses. As discussed before, there
is neither a therapeutic nor preventative vaccine for HSV-1 or HSV-2.
Even for viruses with vaccines available, recent pandemics and epi-
demics have demonstrated the inefficacy of preventative measures
facing mutating variants. With its unique wide-spectrum efficacy,
which has been shown across viruses and variants, the bean powder
gum has the potential to become a standard preventative measure
against various emerging infectious diseases.
RESULTS
In this project, clinical-grade bean powder chewing gum was prepared
using the same formulation and ingredients in the previously approved
IND by the FDA for the ACE2 chewing gum to debulk coronavirus in
saliva of COVID-19 patients (IND 154897, NCT05433181, IRB
851459), replacing the lettuce ACE2 plant powder with lablab bean
powder. Both the drug substance (lablab bean powder) and drug prod-
uct were evaluated for dosage, stability, moisture content, potency, and
bioburden. The potency of FRIL protein and drug product (bean gum)
was evaluated using two different methods (Figure 1). ELISA assay was
used to evaluate aggregation of HSV-1 and HSV-2 by the bean gum to
quantify virus particles remaining in the supernatant after incubation
with the gum extract. Plaque reduction assay was used to evaluate
the infectivity of viruses after incubation with bean gum extract or pu-
rified FRIL protein. Plaque reduction assay was evaluated in four vi-
ruses (HSV-1, HSV-2, H1N1, and H3N2). In potency assays, bean
gums stored between 7 and 794 days at ambient temperature were eval-
uated for stability of functional efficacy.
Stability of FRIL in bean powder up to 683 days
FRIL concentration was first determined in two different stocks of
lablab bean powder stored at 20C freezer for 279 and 445 days.
Optimized ELISA protocol in the lab using primary antibody devel-
oped against FRIL in rabbit and respective secondary antibody was
used for estimation of FRIL in bean powder extracts of both the
stocks. The quantified FRIL in stock-1 (stored 279 days) and
stock-2 (stored 445 days) were 25.07 (0.07) and 24.76 (0.09) mg/mg
of bean powder, respectively (Figure 2A). Less than 5% variability
in FRIL concentration was observed among two different stocks
stored at different time periods. The stability of FRIL in the bean pow-
der of both stocks was further evaluated after additional storage of
238 days. The quantified FRIL in stock-1 (total storage time
517 days) and stock-2 (total storage time 683 days) were 24.49
(0.13) and 23.99 (0.24) mg/mg of bean powder (Figure 2A). During
additional storage of more than 6 months (238 days) only 2.28 and
3.09% reduction was observed in stock-1 and stock-2, respectively.
This confirms the prolonged stability of FRIL in the bean powder.186 Molecular Therapy Vol. 33 No 1 January 2025Overall, less than 5% variability was observed in two different stocks
of bean powder stored, after almost two years (683 days). These ob-
servations confirm the stability and uniformity of FRIL in different
batches of bean powder.
Stability of FRIL in chewing gum up to 790 days at ambient
temperature
Bean gum tablets weremanufactured by Per Os Biosciences (Hunt Val-
ley,MD). Each gumweighs 2000mg and contains 79mg of lablab bean
powder, gum base (24.46%), sugar alcohols - maltitol (15.98%), Xylitol
(1.98%), sorbitol (20.93%) and other compounds, including silicon di-
oxide (0.40%), isomalt (10.00%), stevia 99% (0.45%), natural flavoring
agents, maltodextrin, dextrose, gum Arabic, and essential oils.
We first optimized the procedure of extracting FRIL from the chewing
gum to determine FRIL dosage and stability. FRIL extraction from the
chewing gum was evaluated in two different buffers, PEB and PBS,
with or without Protease Inhibitor Cocktail (PIC) or other protease
inhibitors in the buffer having pH similar to saliva. FRIL quantified
in each bean gum tablet extracted in PEB (with PIC) and PBS
(without PIC) were 1856.63 (89.81) and 1851.90 (24.85) mg per
bean gum tablet, respectively, that reflects efficient extraction of
FRIL from the gum tablet even in the absence of protease inhibitors
and stability of FRIL in the buffer having pH similar to saliva (Fig-
ure S1). The stability of extracted FRIL in both buffers was evaluated
after one cycle of freeze-thawing. Quantified FRIL concentration was
1747.10 (16.37) and 1727.37 (12.43) mg per bean gum tablet in PEB
and PBS, respectively, confirming the stability of FRIL in the freeze-
thawed samples (Figure S1). Two different ratios of buffer are opti-
mized for efficient extraction of concentrated FRIL from the chewing
gum. We obtained FRIL concentration up to 651.55 (15.26) ng/mL of
PEB buffer (Figure S2).
FRIL concentration in the bean gum stored at ambient temperature
for different time periods was determined to evaluate stability. The
FRIL concentration in Protein Extraction Buffer (PEB) was 1871.12
(40.17) mg per bean gum tablet on day 4. FRIL content in gum at
4 days was considered 100% and percentage change was evaluated af-
ter prolonged storage at ambient temperature. Estimated FRIL con-
centrations at 4-, 50-, 286-, 387-, 515-, 655-, 790- and 836-day storage
were 1871.12 (40.17), 1853.86 (7.84), 1859.51 (22.94), 1856.63
(89.91), 1838.06 (35.11), 1776.91 (53.72), 1769.00 (27.11) and
1720.56 (24.40), respectively. Less than 5% variability in FRIL concen-
tration of chewing gum stored up to 790 days at ambient temperature
confirmed stability of FRIL (Figure 2B).
Functional stability of the bean gum
As we observed that FRIL was stable in the bean gum tablets up to
2
years with <5% variability, the functional potency of bean gum stored
at room temperature for the extended period was evaluated using
H1N1 and H3N2 plaque reduction assays (1000 copies of virus per
ml). The previously optimized plaque reduction assay was used for
the newly manufactured (7, 37 days) and older gum tablets stored
at ambient temperature (379, 753, 794, 823 days). For the Influenza
Figure 2. Evaluation of FRIL stability in bean powder
(–20C) and potency of chewing gum stored at
ambient temperature for different durations
(A) FRIL stability in the lablab bean powder analyzed at
279, 445, 517 and 683 days; (B) Stability of FRIL in
Chewing gum analyzed at 4, 50, 286, 387, 515, 655, 790
and 836 days. Proteins were extracted in PEB buffer
(100 mM NaCl, 10 mM EDTA (pH-8.0), 200 mM Tris-Cl
(pH-8.0), 400 mM Sucrose, 2 mM PMSF and 0.5
Protease Inhibitor Cocktail) and FRIL was quantified by
ELISA. Data is represented as mean (standard
deviation), n = 3. FRIL content at earliest time point
considered 100% to compare FRIL content at later time
points; (C) Influenza Virus H1N1 plaque reduction
assays were performed in PBS extracts of bean gum
stored at 7, 37, 379, 753, 794 and 823 days. The
percentage neutralization of the H1N1 was observed at
40.77 mg/mL FRIL-gum. Data is represented as mean
(standard deviation), n = 4.
www.moleculartherapy.orgH1N1 virus, >95% plaque reduction was observed at 40.77 mg/mL of
gum from 7- to 753-day-old gum (Figure 2C). Please see Figure 5
below for more details on functional evaluation of both H1N1 and
H2N3 virus strains. Taken together, the bean chewing gum stored
at ambient temperature is functionally active for up to 2 years.
Evaluation of moisture content and bioburden
Moisture content is one of the crucial parameters in protecting drug
products from contamination and enhance stability upon prolonged
storage. Moisture affects the quality, shelf life, stability and safety of
the drug product as high moisture content facilitates microbial
growth.25,39 To evaluate pharmacopeial compliance, bean powder
and gum tablets were assessed for microbial limits and moisture con-
tent at early (<2months) and long-term storage (
2 years) as per spec-Moleified parameters for orally delivered drugs (Ta-
ble.1). The drug substance lablab bean powder
and bean gum passed the bioburden tests with
no aerobic bacteria as well as yeasts/molds on
trypticase soy agar plates and Sabouraud’s agar
plates, respectively after incubation of 5 days at
30C and (USP<61>). Also, samples were tested
negative for growth of Salmonella and E. coli spp.
as per USP <62>. Bean gum tablets stored at
room temperature for >790 days showed mini-
mal change in moisture content (0.01%). The
absence of viable microorganisms and the mois-
ture content in lablab bean gumpowdermeet the
requirements for clinical-grade drug products, as
stated in a previously approved IND for another
chewing gum product.25,26
FRIL release from chewing gum tablet
To determine the feasibility of topical drug
release from the plant-engineered chewinggum, chewing gum tablets containing the green fluorescent protein
(“GFP gum”) and FRIL (“bean gum”) were studied. Briefly, each
chewing gum tablet contains plant materials with GFP (448 mg) or
FRIL (1856 mg). After simulated chewing, release of target protein
(GFP or FRIL) was quantified. Target protein release from the chew-
ing gums were analyzed at room temperature using a physiologically
comparable model: Artificial Resynthesis Technology (ART-5;
Figure 3A).
The ART-5 is a mastication simulator that mimics human chewing
motions, adapts to food texture changes, and provides immediate,
reproducible computerized feedback.40 FRIL release studies in the
chewing simulator used PEB buffer that includes protease inhibitors
and not human saliva. One criticism could be that proteases presentcular Therapy Vol. 33 No 1 January 2025 187
Table 1. Microbial limits and moisture content analysis of the drug substance (bean powder) and drug product (bean gum)
Test Specification Drug substance Drug product
Storage Age – 45 Days 691 Days 7 Days 790 Days
Storage Conditions Storage Temperature
Lot – 021722
(Stored at 20C)
Lot – 021722
(Stored at 20C)
Lot – 061024
(20C–25C)a
Lot – 041422
(20C–25C)a
(%) Moisture
Content (USP <921>)
<10% 5.8 5.9 1.29 1.28
Microbial Limits
(Bioburden)
(USP <1111>,
<61>, <62>)
Total aerobic microbial
count < 103 CFU/g
0 0 0 0
Total yeast < 103 CFU/g 0 0 0 0
Total molds < 104 CFU/g 0 0 0 0
Absence of Salmonella Absent Absent Absent Absent
Absence of Escherichia coli Absent Absent Absent Absent
aUSP Controlled Room Temperature (20C–25C), Excursions permitted as defined by USP (15C and 30C). Protect from Light.
Molecular Therapyin saliva could degrade FRIL after release. However, quantitation of
FRIL was the same in PEB buffer containing protease inhibitors or
PBS without protease inhibitors (Figure S1). In addition, plaque
reduction assays to evaluate potency were performed using bean
gum extracts in PBS buffer without protease inhibitors. Therefore,
we do not anticipate any changes in release kinetics when bean
gum is evaluated for potency in the clinic.
GFP gum
To optimize release kinetic assays, GFP chewing gums containing
448 mg GFP were studied (data not shown). After 45- and 60- minutes
(mins) of simulated swallowing and chewing, >70% and >90% GFP
was cumulatively released from the GFP chewing gum, respectively
(n = 3). Sustained release of GFP was observed up to 60 min of chew-
ing and continual simulated swallowing.
Bean gum
Topical protein drug release from chewing gums containing 79 mg
lablab bean powder was analyzed (n = 3). During simulated chewing
of the bean gum tablet in the ART-5 mastication simulator, 308.6
(10.7) mg FRIL was released in first 5 min of chewing (16.6% FRIL
release rate). During the following 5 min of chewing, an additional
329.6 (15.0) mg FRIL was released (34.4% cumulative FRIL release),
and after another 5 min of chewing, 353.2 (18.8) mg FRIL was released
(53.4% cumulative FRIL release). The pattern of FRIL release reveals
linearity during the first 15 min of chewing. Interestingly, the next
15 min of chewing released an additional 482.7 (9.8) mg FRIL
(79.4% cumulative FRIL release). The following consecutive 15 min
of chewing released 184.3 (7.5) (89.3% cumulative FRIL release)
and 105.3 (4.9) mg FRIL (95.0% cumulative FRIL release), respec-
tively. Therefore, the FRIL released during the last two cycles of
15-min chewing (from 30 to 60 min chewing) was reduced when
compared to the first 30 min of chewing. Overall, the cumulative
release of FRIL after chewing the gum for 60 min was 1763.5 (47.3)
mg (95.0% release, Figure 3B). Additionally, the proportion of target
protein released from both the GFP and bean gums at 45- and
60-min are comparable, indicating reproducibility of release patterns.188 Molecular Therapy Vol. 33 No 1 January 2025HSV-1 and HSV-2 aggregation by bean gum using ELISA
Potency of the bean gum tablet for HSV-1 and HSV-2 aggregation
was evaluated by incubating the virus with bean gum extract and
detection of virus in the supernatant after centrifugation. The ratio-
nale behind this experimental design is the ability of FRIL to aggregate
viruses.25,33 The bean gum tablets stored at ambient temperature for
352 and 375 days were evaluated for potency against HSV-1 and
HSV-2, respectively. The HSV-1 viruses at 3.75 107 copies/mL titer
were 44%, 69%, and 75% aggregated at bean gum concentrations of
33.5, 100.5, and 167.5 mg/mL respectively. While the HSV-2 viruses
at 3.1  106 copies/mL titer were 47%, 58%, 77%, 80%, and 94%
aggregated at bean gum concentrations of 16.7, 33.5, 100.5, 167.5,
and 335.0 mg/mL respectively. Slightly higher percent aggregation
for HSV-2 compared to HSV-1 was observed, when compared at
similar bean gum concentrations (Figure 4). Considering an average
1.1 mL of saliva before swallowing in humans,40,41 the chewing of one
2000 mg gum tablet would be 
6 and 
12 times more efficacious to
achieve 94% and 75% aggregation of HSV-2 and HSV-1, respectively
in the oral cavity. This result reflects the clinical relevance of the uti-
lization of bean gum to prevent infection and transmission of HSV-1
and HSV-2 viruses.
HSV and influenza virus neutralization by the bean gum using
plaque reduction assay
As FRIL has binding affinity to complex-type N-glycans, the presence
of several complex-type N-glycans on HSV glycoproteins lays the sci-
entific foundation of potential antiviral efficacy of FRIL against HSV,
H1N1 and H3N2 (Figure 1). Certain components of bean gum inter-
fere with the plaque reduction assay based on the viral strain and cell
line used. Therefore, the placebo bean gum was evaluated first in pla-
que reduction assays. The placebo gum is identical to bean gum in
appearance, taste, smell and composition except that it doesn’t
contain the bean powder. Dialysis of gum extract to remove soluble
components didn’t change the outcome of H3N2 plaque reduction as-
says usingMDCK cells (Figures S3A and S3B). However, the presence
of gum components reduced the number of plaques (10–20%) in
HSV-1 plaque reduction assay using Vero cells at highest
Figure 3. ART-5 mastication simulator and
quantitation of total protein and FRIL released from
bean gum using ART-5 mastication simulator
Overview of ART-5 mastication simulator (A) Full view;
(B) top view, (C) Modified third human molars; (D)
Chewing of the bean gum tablet. (E) Protein release
was quantified at 0-, 5-, 10-, 15-, 30-, 45-, and 60-min
of simulated chewing in PEB buffer (100 mM NaCl,
10 mM EDTA (pH-8.0), 200 mM Tris-Cl (pH-8.0),
400 mM Sucrose, 2 mM PMSF and 0.5 Protease
Inhibitor Cocktail).Total proteins quantified by the
Bradford assay and FRIL was quantified by ELISA.
Chewing data were analyzed by a one-way ANOVA test
and is represented as mean (standard deviation), n = 3,
change in release between 0 and 60 min is significant =
****p-value <0.00001.
www.moleculartherapy.orgconcentrations evaluated (Figure S3C). Therefore, all H1N1 and
H3N2 plaque reduction assays were performed in gum extracts
without dialysis (Figure 5). HSV-1 and HSV-2 plaque reduction as-
says using Vero cells were performed using dialyzed gum extracts
to exclude interference of gum components (Figure 6). Therefore,
any impact of components on plaque reduction assays were addressed
by their removal through dialysis. All plaque reduction assays con-
tained 
1000 copies/mL of tested virus. For H1N1, >95% plaqueMolereduction was observed at 40.77 mg/mL of
bean gum from 7- to 753-day-old gum
(Figures 5A–5D). H3N2 was assessed at two
time points: 
1 year (410-day) and 
2 years
(823-day) old gum. Over 95% neutralization
of H3N2 was observed at 40.77 mg/mL of
2-year-old gum and close to 95% neutralization
was observed at 40.77 mg/mL of 1-year-old
gum (Figures 5F and 5G).
As the volume of saliva before swallowing in
humans is an average of 1.1 mL,41,42 we estimate
that 2000 mg bean chewing gum tablet (
50
of 40.77 mg/mL needed to reach a clinically sig-
nificant reduction in viral infectivity) is suffi-
cient to completely neutralize the two Influenza
virus strains in the oral cavity. Similarly, for
HSV-1 and HSV-2, we estimate the 2000 mg
gum tablet in 1.1 mL saliva is 
12 and

27 of concentration needed to reach a clin-
ically significant reduction of viral load, which
gives FRIL potential to be a highly efficacious
anti-viral solution for oral cavity.
Antiviral potency comparison of bean gum
with purified FRIL
In order to distinguish antiviral effect of gum
components from FRIL present in the bean
gum, we evaluated their impact independently.Purified FRIL protein showed similar level of inhibition potency
with >10 mg/mL (1mg purified protein used in 100 mL treatment vol-
ume) neutralizing >90% of Influenza virus H1N1 and 95% of Influ-
enza virus H3N2 (Figures 5H and 5I). The bean gum required
40.77 mg/mL containing 36.07–38.14 mg/mL of FRIL to achieve
similar neutralization of Influenza viruses (Figures 5A–5F). Addi-
tionally, bean gum neutralized >95% of HSV-1 virus at
148 mg/mL FRIL in 159.84 mg/mL bean gum (Figures 6A andcular Therapy Vol. 33 No 1 January 2025 189
Figure 4. HSV-1 and HSV-2 aggregation at different
concentrations of the bean gum extract
Virus strains were incubated with different concentrations
of bean gum extract (mg/mL) for 1 h, and after centrifu-
gation viruses in the supernatant were quantified by
ELISA. Proteins were extracted in PEB buffer (100 mM
NaCl, 10 mM EDTA (pH-8.0), 200 mM Tris-Cl (pH-8.0),
400 mM Sucrose, 2 mM PMSF and 0.5 Protease
Inhibitor Cocktail). Virus titer used for HSV-1 and HSV-2
were 3.75  107 pfu/mL and 3.1  106 pfu/mL,
respectively. Data is represented as mean (standard
deviation), n = 3.
Molecular Therapy6B) compared to 68.52 mg/mL FRIL contained in 74.00 mg/mL bean
gum against HSV-2 virus (Figures 6C and 6D). Over 95% neutral-
ization of HSV-1 and HSV-2 viruses were observed when virus are
co-incubated with purified FRIL protein at 80 mg/mL for HSV-1
(8mg purified protein used in 100 mL treatment volume) and
20 mg/mL for HSV-2 (Figures 6E and 6F). Requirement of different
doses of FRIL to neutralize HSV-1 and HSV-2 is likely due to
different glycosylation patterns in the glycoproteins that are key
to mediating cellular entry. Several glycoproteins, such as gC and
gG, are involved in the viral entry process and are absent in
HSV-1.38,43190 Molecular Therapy Vol. 33 No 1 January 2025This comparison between bean gum and puri-
fied FRIL shows that the antiviral efficacy we
observed is mainly from FRIL protein and addi-
tional anti-viral effect from other gum compo-
nents is minimal, as higher concentration of
FRIL is needed against both HSV and Influenza
viruses. The difference between the amount of
purified FRIL protein needed to reach >95%
neutralization can be attributed to incomplete
release of FRIL protein from the bean gum in
plaque neutralization assay (incubation without
stimulating release). However, 2000 mg bean
chewing gum containing 1800–1900 mg FRIL
should be sufficient to neutralize virus in the
oral cavity. Compared to purified FRIL that re-
quires expensive purification and cold storage,
bean gum is a better choice for anti-viral treat-
ments as it is functionally stable for up to 
2
years when stored at ambient temperature.
Lablab bean powder safety evaluation
The presence of vicine and convicine (v-c) in
lablab bean powder and chewing gum was
investigated. We used faba bean (Vicia faba
L.) as a legume species with high v-c levels in
comparison to lablab products (Figure S5). A
faba bean seed sample (accession ILB 938\2)
with a high concentration of v-c was used in
HPLC analysis in comparison to lablab bean
samples. The total concentration of v-c in fababean seed was 1.05% of seed dry matter (10,470 mg/kg). No v-c
was detected in either lablab bean powder or chewing gum, ensuring
the safety of the chewing gums.
DISCUSSION
There is no vaccine for HSV, but it is themost commonly transmitted,
impacting 27% of American adults.44 Unlike other viruses where
symptoms are readily evident, HSV is shed and transmitted asymp-
tomatically; HSV-1 accounts for >50% of new infections among col-
lege students and heterosexual women.44,45 HSV-1 is universally
prevalent in Africa, with an increase in seroprevalence for several
Figure 5. Functional evaluation of bean gum
Potency evaluation of Influenza virus strains H1N1 and H3N2 using bean gum tablets stored at ambient temperature for different time points: Percent neutralization of H1N1
at (A) 7-days; (B) 37-days; (C) 379-days; (D) 753-days; (E) 794-days. Percent neutralization of H3N2 at (F) 410-days; (G) 823-days. The red line shows 95% neutralization.
Purified FRIL evaluated against H1N1 (H) and H3N2 (I) is also shown for comparison. All gum extracts were prepared in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, and 1.8 mM KH2PO4, pH-7.4). All assays were performed using viral titer of
1000 copies/mL. Data is represented as mean (standard deviation), n = 4 for H1N1
and n = 6 for H3N2.
www.moleculartherapy.orgdecades, mostly through oral-to-oral transmission.45 HSV-2 seropre-
valence is high among HIV patients (65% in the US, 85% in Africa
among heterosexuals)46; unfortunately, anti-retroviral therapy
doesn’t reduce HSV-2 acquisition47 and HSV has already developed
resistance to widely used antiviral drugs.48 As reported by Moin
et al.,47 HSV prevalence is high throughout the globe: 55–67% of
the population (28% had oral lesions) in Spain, Germany, and France;
In Asia, much higher HSV-1 seroprevalence was observed in India,
China, and Japan ranging from 88% to 96%; In Africa (Egypt and
Morocco) the number is 98–99%, and in South America 63 to 83%
for HSV-1 and HSV-2; In Australia, HSV-1 infection increased
16% in 2017 from 45% in 2004.
Herpes labialis is the most common orofacial form of infection,
known as cold sores, and HSV is primarily transmitted through le-
sional fluids, saliva, and even though brief kissing from a father to a
child.47–49 Although HSV-2 was believed to be not primarily trans-mitted orally, due to increase in oral sex prevalence, HSV-2 shedding
through oral cavity is now more common among the general popula-
tion. Although HSV-2 is the leading cause of recurrent genital herpes,
genital HSV-1 is now known to affect millions of individuals in the US
and is currently the leading cause of newly diagnosed genital herpes
infection in the US.50 Recently analyzed saliva samples of 220 individ-
uals revealed that HSV-2 is more commonly shed in the oral cavity
than HSV-1.51 However, HSV-2 oral shedding has been documented
for several decades among HIV-positive men, again due to oral sex.52
HSV-2 shedding through oral mucosal lesions has also been docu-
mented for several decades.53 These statistics underscore the need
to reduce transmission of HSV.
Although lack of vaccine is pointed out as a limitation for HSV, most
recent vaccines confer protection from the severity of the disease but
do not prevent infection or transmission. This controversy was widely
debated in the public press during the recent COVID-19 pandemic.Molecular Therapy Vol. 33 No 1 January 2025 191
Figure 6. Potency evaluation of bean gum tablet using HSV-1 and HSV-2
HSV-1: Over 95% neutralization observed at 159.84 mg/mL bean gum containing 148.00 mg/mL FRIL; Experiments performed with (A) 325-day (B) 413-day old gums. HSV-
2: Over 95% neutralization observed at 74.00 mg/mL bean gum containing 68.52 mg/mL FRIL; Experiments performed with (C) 325-day (D) 413-day old gums. Purified FRIL
evaluated against HSV-1 (E) and HSV-2 (F) is also shown for comparison. The red line shows 95% neutralization. All gum extracts were prepared in PBS buffer (137mMNaCl,
2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH-7.4), and dialyzed in PBS buffer overnight to reduce the concentration of sugar alcohols and other inferring
compositions of bean gum. All assays were performed using viral titer of 
1000 copies/mL. Data is represented as mean (standard deviation), n = 4 for bean gum against
HSV-1 and HSV-2, n = 2 for purified FRIL protein against HSV-1 and HSV-2).
Molecular TherapyFully vaccinated individuals have breakthrough COVID-19 infec-
tions, and the peak viral loads are similar to unvaccinated individuals
and efficiently transmit viruses in household settings.6 The scientific
explanation is that recent vaccinations confer systemic immunity that
predominantly produced IgG and not secretory IgA (mucosal immu-
nity) that neutralizes viruses at their points of entry. Global use of oral
polio vaccine that offers both mucosal and systemic immunity has
demonstrated the importance of vaccines to prevent transmission,
in addition to conferring protection.54 All current Influenza vaccines
develop systemic immunity and, therefore, do not prevent transmis-
sion. Airborne volume of saliva droplets in healthy subjects is 3–5 or-
ders of magnitude higher than breath droplets and speaking four
words transmits more virus than 1 h of maskless breathing.55 There-
fore, in this study we evaluate the ability of the antiviral protein to
reduce viral load delivered using chewing gum, to facilitate slow
release of antiviral protein for better efficacy in the oral cavity.
To facilitate the evaluation of bean gum in human clinical trials, we
prepared clinical-grade drug product and characterized them to
meet regulatory requirements. FRIL is stable in the ground bean pow-
der when stored at 20C up to 683 days. Storage of powder is not
necessary because lablab been seeds could be ground from seeds192 Molecular Therapy Vol. 33 No 1 January 2025whenever required. However, to meet regulatory requirements,
drug substance (bean powder) was stored to evaluate dosage and sta-
bility. During this process, we optimized different buffer conditions
and protective agents, including protease inhibitors because plant
cells release abundant proteases in crude extracts. Fortunately, none
of these stability agents were required because FRIL was stable in
PBS buffer without any additives. The stability of FRIL in plant pow-
der is not unique because we have previously observed stability of hu-
man, bacterial, or fungal proteins in freeze-dried plant cells for several
years when stored at ambient temperature.19–26,56,57
FRIL is stable when gum is stored at ambient temperature for up to
790 days. This observation is not surprising because we have observed
such long-term stability of proteins in several other chewing gums
products (up to 3 years in GFP chewing gum).21 Bean gums used
for Plaque reduction assay were stored for 7–823 days at ambient
temperature. Chewing gums contain the same ingredients and per-
centages as the gum in previously approved IND (gum base, sugar al-
cohols, components). The drug substance lablab bean powder and
bean gum passed the bioburden test with no aerobic bacteria as
well as yeasts/molds, following USP <61>, <USP 62> guidelines for
evaluation, like previously approved IND requirements. The moisture
www.moleculartherapy.orgcontent for both bean powder and bean gum tablet is 5.9% and 1.28%
respectively, which is within the acceptable range of <10%. The
absence of viable microorganisms and moisture content in lablab
bean powder and the bean gum powder meets requirements for clin-
ical-grade drug products, based on previously approved IND for
another chewing gum product.25
Mimicking that of human swallowing, the ART-5 continually re-
moves and introduces liquids and proteins while chewing, suggesting
that results are physiologically relevant. Since the bean gum showed
steady and significant release FRIL, the bean gum is suitable for eval-
uation of self-infection as swallowing saliva is critical to neutralizing
viruses in the throat surface. Moreover, the majority of FRIL is
released from the chewing gum tablets after only 15 min of simulated
chewing (53.4% cumulative FRIL release). Complete inhibition of

1,000 copies/mL H1N1, H3N2, HSV-1, and HSV-2 viruses are
observed at 38.14 mg/mL, 38.14 mg/mL, 148 mg/mL, and
68.5 mg/mL of FRIL protein, through the plaque reduction assays.
As discussed earlier, 
475 mg/mL FRIL was released within the first
5 min of chewing. While the potency of bean gum could vary depend-
ing on virus variants and the viral titer in the patient’s oral cavity
differs from individual to individual, we believe significant neutraliza-
tion could be achieved within 5 min of chewing. Additionally,
continual FRIL and total protein released at each time point, despite
continuous removal of solution, highlights the feasibility of sustained
topical FRIL delivery while chewing (Figure 3E). Such observation
lays the foundation clinical evaluation.
Mechanistically, FRIL has been shown to exhibit a homogeneous
tetramer structure with four carbohydrate-binding domains (CBD).
The four CBDs are identical and facilitate binding of FRIL to com-
plex-type N-glycans.32,33 In addition, FRIL facilitates crosslinking to
form higher-order structures through protein-protein interactions
like van der Waals interactions.33 Therefore, FRIL interaction with
complex-type N-glycans, through virus binding and self-binding to
form large aggregates of entrapped viruses prevents virus from
entering the host cell and escaping from the endosome upon entry.
The potency of bean gum was evaluated using two different methods.
ELISA was used to evaluate aggregation of HSV-1 andHSV-2 by FRIL
to quantify virus particles remaining in the supernatant after incuba-
tion with the bean gum extract. The total volume of saliva droplets is
several orders of magnitude higher than breath droplets.9 Therefore,
trapping virus particles in chewing gum is a key step in prevention of
oral virus transmission. Bean gum extracts trapped HSV-1 up to 75%
at virus titer 3.75  107 pfu/mL and HSV-2 up to 94% at virus titer
3.1  106 pfu/mL, in a dose-dependent manner. ELISA measures
only virus aggregation but plaque reduction assay measures both viral
aggregation and blockage of H1N1, H3N2, HSV-1, HSV-2 at the en-
dosomes by FRIL in host cells.25,32 Therefore, lower inhibition
(
75%) is observed in ELISA whereas >95% inhibition is observed
in plaque reduction assays. In addition, percentage inhibition be-
tween these assays is not comparable because plaque reduction assays
were done with 1,000 viruses per ml (amount reported in the oral cav-ity), whereas ELISA assays are done with 1–10 million viruses per ml
in order to increase absorbance signal.
Therefore, plaque reduction assay was used for evaluation of aggrega-
tion and blockage of virus not trapped by FRIL at the endosomes in-
side cells after entry.32 This assay shows not only aggregation to trap
viruses but also the prevention of infection. Therefore, plaque reduc-
tion assay was evaluated in four viruses (HSV1, HSV-2, H1N1,
H3N2). Virus neutralization (>95%) was observed at 
40 mg/mL
of bean gum with H1N1 and H3N2, when 
1000 copies/mL of vi-
ruses were used in plaque reduction assays. Influenza virus titer in in-
fected patients beyond 2 days after symptom onset, as measured by
viral RNA concentrations, is reported to be 3.62 (2.13) log10 copies/
mL.58 Because each gum tablet weighs 2000 mg and only 
40 mg/
mL is required for >95% neutralization, bean gum tablet has
50-fold higher than efficacy dose and therefore it is suitable for eval-
uation in the clinic.
For HSV-1, 160mg of bean gum resulted in >95% inhibition of plaque
formation without negative impact on cell proliferation, in dialyzed
gum extracts to remove interference from components. For HSV-2,
>95% inhibition of plaque formation was observed with 74.00 mg/
mL. Differential neutralization efficacy between HSV-1 and HSV-2
is most likely due to different glycosylation patterns of surface glyco-
proteins. For example, Glycoprotein G is glycosylated in HSV-2 but
not in HSV-1.59 Higher glycosylation of HSV-2 may correlate with
lower dose of FRIL required for neutralization, but further mecha-
nistic studies are needed. For HSV-1 virus, viral DNA titer greater
than 2.3 log10 copies/mL is generally considered positive. For
HSV-2 virus, quantities greater than 4 log10 DNA copies/mL have
been modeled as leading indicator to interpersonal transmission.17
Considering errors that could occur from RT-PCR measurement,
we believe our experiment design provides a close simulation to viral
titer of infected patients’ saliva. Each bean gum tablet (2000 mg) has
27-fold for HSV-2 and 12.5-fold higher for HSV-1 neutralization ef-
ficacy and is therefore suitable for clinical evaluation.
HSV-1 KOS strain used in our study is a clinical isolate collected from
Kendall O. Smith, a well-known virologist at Baylor College of Med-
icine, Houston TX.60 KOS 63 and variants are of Asian origin and
KOS 79 strain is common in North America and Europe.61 HSV –
strain 333 also originated from Texas.62 Both these strains are exten-
sively used in various preclinical and clinical investigations. While
monovalent glycoprotein D conferred protection against HSV chal-
lenge,63 clinical evaluation utilizes trivalent mRNA vaccine contain-
ing three different glycoproteins.64 In our virus trap investigations,
ELISA assays used antibodies against both B2 and D2 glycoproteins,
confirming that FRIL interacts with both these glycoproteins, which
are common to most HSV clinical strains and play a critical role in
viral entry into host cells.65 Dose dependent reduction by FRIL shows
efficient binding to highly conserved glycoproteins, thereby predict-
ing efficacy against several clinical strains despite their variability
among other surface proteins.Molecular Therapy Vol. 33 No 1 January 2025 193
Molecular TherapyLablab bean powder is Generally Regarded as Safe (GRAS notice No
GRN 000879, 2020) by the FDA based on toxicology reports submit-
ted.66 Genetic diversity among Lablab purpureus, faba, or fava beans
is minimal. Evaluation of simple sequence repeat markers shows high
similarity among different beans, showing their common genetic
ancestry.67 The primary difference is the presence of glucosides vi-
cine/convicine in Faba beans that could be harmful to glucose-6-
phosphate dehydrogenase (G6PD) -deficient patients causing
favism.68,69 However, lablab bean has no detectable levels of vicine/
convicine (Figure S4), and therefore is safe for consumption.
Lablab is a versatile crop. Lablab dry seeds are prized for their nutri-
tional value: rich in protein, fiber, vitamins, and minerals.70 In Afri-
can and Asian countries, Lablab consumption is promoted to address
malnutrition.71–74 It originated in tropical Asia and Africa and was
cultivated in India as early as 1500 BC.75 Its consumption as food
spans centuries and continents, with its origins traced back to Africa,
particularly around Ethiopia, where it was first cultivated for its edible
seeds and pods. From there, it gradually spread to other regions,
including tropical Asia. Today, lablab is cultivated in various tropical
and subtropical regions worldwide, including parts of South America
and Australia.76
The safety and efficacy of bean powder have been studied in several
animal models. Intranasal administration of 2.9 mg or 29 mg/kg/
day purified FRIL was well-tolerated and therapeutic when adminis-
tered to H1N1-infected mice.32 Several safety studies have been con-
ducted using bean powder in mice and rats. IP administration of
30 mg/kg (60 above the gum dose) was well-tolerated in a mouse
tumor model.77 As reported in the GRAS determination,66
Schmandke et al. conducted 4-week feeding studies of several
different processed bean powder formulations in male and female
Shoe:Wist/Il rats (30 rats per group, dosage 20, 40, and 80 g/kg).78
The low dose of 20 g/kg was considered the no-observed-effect level
(NOEL). Compared to the gum, lablab bean dose of 
0.0007 g/kg in
humans, this rat NOEL is more than the target clinical dose by several
orders of magnitude. The dose of lablab bean powder in the bean gum
is 79 mg, when compared to 500 g ingested in human clinical toxi-
cology evaluation.69 This is 6,330-fold less than what would be
consumed as food. Chewing gum has evolved over 9000 years, tran-
sitioning from chewing resin and sap directly from trees to medicated
gums containing aspirin, nicotine, or vitamins.79
Interestingly, FRIL preserves hematopoietic progenitors80 and neural
progenitors,81 and has been used to prolong in vitro maintenance of
quiescent human cord blood CD341CD382/low/SCID repopulating
stem cells.82 Cells cultured with FRIL engraft differentiated into
erythroid myeloid and lymphoid lineages in the bone marrow of
both primary and secondary transplanted recipients. Unlike other
plant lectins, FRIL does not induce cell proliferation or IL-6
secretion.82
Seasonal Influenza viruses expelled from the oral cavity contribute to
viral transmission in both low- and high-humidity environments.83194 Molecular Therapy Vol. 33 No 1 January 2025Several antiviral products have been evaluated in the clinic to reduce
viral load and symptoms. Iota-carrageenan is a polymer derived from
seaweed and traps viruses.84 Although initial studies reported positive
results,85 reanalysis of randomized trial data of Carrageenan nasal
spray showed a recovery rate of 54%, much lower than >200%
observed for Zinc lozenges in five different clinical trials.86
Echinaceae/Salvia lozenges derived from plants showed a reduction
in viral load but required very high doses - 16,800 mg per day plant
extracts, resulting in higher adverse events.87,88 Nasal spray of Iota-
carrageenan in COVID-19 patients showed significant efficiency in
preventing COVID-19 in healthcare workers managing patients
with this disease.89 Careful evaluation of Zinc or vitamin C in
COVID-19 patients had negligible impact on infection.90 Lozenges
containing amylmetacresol, 2,4-dichlorobenzyl alcohol or hexylresor-
cinol had no measurable antiviral activities.84 Furthermore, broad-
spectrum antiviral chemicals result in viruses developing resistance,
which is especially a major challenge to control HSV.91 Several
mouthwashes with virucidal effects were reported.92 Several sprays
containing antiviral products were marketed as OTC products during
COVID-19 pandemic, but the FDA published warning notices for not
providing proof for clinical efficacy for those products. Therefore,
there is a great need to develop efficacious oral topical antiviral drugs.
FRIL protein is efficacious at very low concentrations by trapping vi-
ruses like broad-spectrum chemicals but also enters human cells and
blocks viruses at the endosomes. Chewing gum as a delivery vehicle is
preferable to lozenges because of slow and prolonged release of drug
substances79 but direct comparative studies of delivery methods using
the same antiviral product are needed. Clinical evaluation of different
delivery methods (lozenges, spray, tablets, drops) didn’t result in
measurable outcomes, although authors recommended orally deliv-
ered formulations.88
A phase 3 study to assess the efficacy of the antiviral drug baloxavir to
evaluate household transmission of Influenza is in progress
(NCT03969212).93 A very large study in 208,225 families with partic-
ipants in different age groups showed that oral antiviral delivery is
better than inhalers to control household viral transmission.93 Evalu-
ation of different neuraminidase inhibitors showed that household
transmission was highest with oseltamivir.94 Although Peramivir or
Zanamivir neuraminidase inhibitors are effective, they are delivered
intranasally or intravenously.95,96 However, our study focuses on
oral delivery. Oseltamivir is the most widely used anti-Influenza
drug globally and is orally delivered.96,97 A detailed analysis of 2247
citations on non-pharmaceutical interventions (hand washing, disin-
fection, oral hygiene, physical barriers, etc.) did not reveal any conclu-
sive benefits.97 Topical delivery of biologics to control virus infection
and transmission is an emerging new area of research.79,98 Chewing
gums containing antiviral proteins are efficient in significantly
(>90%) reducing viral load of several SARS-CoV-2 strains (Beta,
Delta, Omicron) in saliva samples of COVID-19 patients.25,26 Similar
ex vivo clinical studies were not feasible for influenza or Herpes
patients because ongoing vaccine studies do not evaluate mucosal
samples. Injectable vaccines confer protection through IgG and not
secretory IgA that could block viruses at their points of entry and
www.moleculartherapy.orgprevent transmission.54,99,100 Chewing gums containing enzymes
disrupt the dental plaque and kill bacteria and yeast that cause dental
caries.22 Preparation and CMC characterization of bean gum and po-
tency to neutralize HSV1, HSV-2, H1N1 and H3N2 viruses in this
study augur well for advancing this product to the clinic to minimize
infection or oral transmission.
MATERIALS AND METHODS
Estimation of FRIL in lablab bean powder
We received lablab (hyacinth) bean (Lablab purpureus (L.) Sweet)
seed powder from Dr. Che Ma (Genomics Research Center,
Academia Sinica, Taiwan) and estimated FRIL concentration as
described earlier32 with some modifications. Lablab bean seed ground
powder (100 mg) suspended in 1 mL of protein extraction buffer
(PEB, 100 mM NaCl, 10 mM EDTA (pH-8.0), 200 mM Tris-Cl
(pH-8.0), 400 mM Sucrose, 2 mM PMSF and 0.5 Protease Inhibitor
Cocktail), was mixed properly through vortexing, and incubated for
1 h at 4C on homogenizer (Vortexer). The solution is then sonicated
6 cycles (10 s on and 15 s off) at 80% amplitude and spun at 750  g
for 5 min at 4C. Supernatant was collected and quantity of soluble
FRIL (Flt3 Receptor Interacting Lectin) was estimated by ELISA. In
ELISA, 100 mL of bean powder extract supernatant or purified FRIL
protein as standards was coated in the wells of microtiter plate (Corn-
ing Incorporated, Costar Assay Plate 3590, NY, USA) and incubated
overnight at 4C. Next day, after washing with PBST (PBS +0.05%
Tween 20) four times, the blocking was done with PBST having 3%
non-fat dry milk for 1 h at 37C. After three washes with PBST, the
plate was incubated with 100 mL of anti-FRIL (primary antibody
developed in rabbit) antibody (1:2000), at 37C for 1.5 h. Plate was
then washed three times with PBST, and incubated with 100 mL of
anti-Rabit IgG raised in goat conjugated with HRP (1:5000), incu-
bated at 37C for 1.5 h. Finally, plate was washed three times with
PBST and 100 mL of HRP substrate (TMB/E Solution, Mllipore
Carp.) was added in each well. The plate was incubated for 2 to
10 min at RT depending on developed color intensity. Reaction was
stopped by adding 50 mL of 2N H2SO4 and absorbance measured at
450 and 700 nm using microplate reader (BioTek, Synergy H1, VT
USA). Additional absorbance at 700 nm measured to normalize
any possible absorbance due to the turbidity of the extracts. FRIL pro-
tein was estimated by comparing standards. Experiments were per-
formed in triplicate and data presented as mean (standard deviation).
Chewing gum preparation from the lablab bean powder
Chewing gum tablets containing ground lablab bean seed powder
were prepared by Per Os Biosciences (Hunt Valley, MD), via
compression process, allowing for homogeneous availability of pro-
tein drugs without raising the temperature. The bean gum tablets
were prepared with the following excipients – gum base (24.46%),
sorbitol (20.93%), maltitol (15.98%), Xylitol (1.98%), magnesium
stearate (3.00%), silicon dioxide (0.40%), isomalt (10.00%), stevia
99% (0.45%), natural flavoring agents (dextrose, maltodextrin, essen-
tial oils, gum arabic) to make the gum tablets flavorful, and conducive
to compression. The physical architecture and flavor of the prepared
bean gum tablets were similar to other non-therapeutic chewing gumsavailable in the market made by conventional methods that use high
temperature and extrusion. The received gum tablets from Per Os
Biosciences were stored in the mylar bags to avoid moisture absorp-
tion and light exposure. Few tablets were kept in the Uline black con-
tainers for routine examination viz; bioburden, moisture content,
drug dose quantitation and release.
Estimation of FRIL in the prepared chewing gum
Estimation of FRIL in the prepared chewing gum of lablab bean pow-
der was done after extracting total soluble proteins in two different
buffers (i.e., Phosphate Buffer Saline (PBS) and PEB. A bean gum
tablet of 2000 mg was crushed and ground into the mortar and pestle.
Afterward, 334 mg grinded material was suspended in 1 mL of PBS or
PEB, mixed properly, and incubated for 1h at 4C on homogenizer
(Vortexer). Samples were then sonicated 9 cycles (10 s on and 15 s
off) at 80% amplitude and spun at 750  g for 5 min at 4C. The su-
pernatant was then collected, and soluble FRIL quantified by ELISA.
Following the same protein extraction and ELISA protocol, stability
of FRIL in chewing gum extracts stored in freezer and single time
thawed were determined. Experiments were performed in triplicates
and data represented as mean (SD), n = 3.
Protein release from GFP and bean gum tablets
The feasibility of protein-impregnated chewing gum tablets for
topical application was evaluated by the protein released after simu-
lated chewing and swallowing through the ART-5 mastication simu-
lator. To replicate swallowing while chewing within the oral cavity,
the ART-5 was programmed to continually introduce and remove liq-
uids at an average flow rate (PEB introduced) of around 100 mL (mL)
per minute of chewing. The angle of bite ranged between 0 and 13,
resulting in an extension of 11.6 mm. Similar to human chewing
speeds,101 an average of 1.00 Hz (Hz) was coded into the chewing
software. To facilitate physiologically comparable results, the
ART-5 was modified to include sound upper and lower 3rd human
molars. After taking acrylic impressions, the extracted molars were
ground to 2 mm (mm) of enamel on occlusal surfaces of the maxilla
and mandible and mounted into the ART-5 using resin (Figure 3A).
To improve the ability of the 2-g chewing gum tablets to stick to the
sound human molars in the ART-5 machine during early chewing,
tablets were pre-soaked in 500 mL PEB buffer for 30 min. Over this
30-min interval, >90% PEB was absorbed by the chewing gums,
and total protein released from the chewing gum tablets from this in-
cubation step was minimal (<5%). The pre-soaked chewing gum tab-
lets were then placed onto the mandible of the ART-5 machine, with
simulated swallowing and chewing motions commencing afterward.
The buffer swallowed after chewing was collected and stored on ice
at 0-, 5-, 10-, 15-, 30-, 45-, and 60-min. Protein released from the
chewing gums were then analyzed at the aforementioned timepoints.
Release from the GFP gumwas quantified by using known concentra-
tions of the recombinant GFP protein (Cell Biolabs STA-201). For the
GFP chewing gums, the samples retrieved after simulated swallowing
were then loaded into a 96-well black plate (Corning Incorporated,
Costar Assay Plate 3916, ME USA). The fluorescence intensity wasMolecular Therapy Vol. 33 No 1 January 2025 195
Molecular Therapyread on a multi-mode plate reader (BioTek, VT) at 485 nm (excita-
tion) and 538 nm (emission). For the bean gums, the released FRIL
was quantified using ELISA as described previously using the anti-
FRIL antibody as the primary antibody and its respective secondary
antibody.
HSV-1 and HSV-2 aggregation with bean gum extracts
In HSV aggregation assay, bean gum protein extract of different con-
centrations in protein extraction buffer (100 mM NaCl, 10 mM
EDTA, 200 mM Tris-cl (pH-8), 400 mM Sucrose, 2 mM PMSF and
0.5 PIC) incubated with Virus (HSV-1 and HSV-2 titer were
3.75  107 pfu/mL and 3.03  106 pfu/mL, respectively) in presence
of 100 mM MnCl24H2O for 1h at 37C with intermittent mixing.
FRIL supernatant without virus and virus without FRIL supernatant
used separately as control. After incubation, spun at 14000 rpm for
10 min. Supernatant collected and evaluated for the presence of virus
by the ELISA. In ELISA assay, 50 mL of supernatant coated into wells
of ELISA plate and incubated for 3 h at RT. After three washes with
PBST, blocking was done with 200 mL of 5% non-fat dry milk in PBST
overnight at 4C. Wells washed three times and incubated with pri-
mary antibodies (anti-glycoprotein B and anti-glycoprotein D of
HSV-1 or HSV-2 raised in rabbit, mixed, each 5 mg/mL) and incubate
for 2 h at RT. After three washes secondary antibody (goat anti-rabbit
IgG conjugated with HRP) added and plate incubated for 2 h at RT.
After three washes with PBST, 100 mL of substrate (TMB/E Solution,
Mllipore Carp.) added and incubate for 2–10 min at RT for color
development. The reaction was stopped by adding 50 mL of 2 N
H2SO4, and absorbance measured at 450. After subtracting the absor-
bance of respective controls, percent of virus remained into the FRIL
treated sample was calculated. Accordingly, inhibition was calculated.
Aggregation experiment performed in triplicates and data repre-
sented as mean (SD).
Moisture content analysis by Karl Fisher coulometry
Moisture content assessment was done as per USP <921> Method I
(coulometry). Titrando 860 kF Thermoprep and Titrando 851 were
used and the titration parameters viz; temperature and pump flow
rate were set to 150C and 50 mL/min on the Titrando 860 kF Ther-
moprep respectively. The instrument was monitored for constant
drift of 10.0 (5.0) mg/min before performing any estimations. This
is the important step for precise measurement of moisture content.
To measure the residual water content of moisture present in the
empty glass vial, it was sealed with aluminum cap and used for titra-
tion. Three such titrations were performed to measure blank to get
averaged value for final calculation. For measurement of standard,
50.0 (2.0) mg of HYDRANAL-water standard powder was weighed
in glass vial (Cat. Code 34693, Honeywell Specialty Chemicals Seelze
GmbH, KF Oven 150C–160C) and capped with aluminum stopper
immediately. Two such vials were prepared and assessed for moisture
content. The exact weight of water standard was recorded in the pa-
rameters section of the KF machine. The instrument was calibrated
using blank in triplicates and water standard in duplicates. The stan-
dard should be in the range 4.96–5.04% for hydranal at the specified
parameters. For measuring sample, bean gum tablet was homoge-196 Molecular Therapy Vol. 33 No 1 January 2025nized in mortor pestle and 50 mg of the gum tablet powder was
weighed in glass vial stoppered with aluminum seal and bottle was in-
serted on KF coulometer for moisture content. Similarly, 50mg lablab
bean powder was weighed in glass bottle, capped with aluminum seals
and analyzed for moisture content.
Bioburden assessment
The lablab bean powder and bean gum powder were analyzed for bio-
burden test was done as per United States Pharmacopeia guidelines
USP <61>, <62> to assess aerobic bacterial and yeast/mold counts
and presence of pathogenic bacteria viz; Salmonella and E.coli respec-
tively. For aerobic bacterial and yeast/mold counts sterile Trypticase
Soy Agar (TSA- Becton Dickinson) and Sabouraud’s Dextrose agar
plates (SDA - Oxoid) were used respectively. Bean powder was
directly used for preparation of suspension for microbial enumera-
tion. bean gum tablet was transferred aseptically to pre-sterilized
mortar and pestle and homogenized to obtain uniform powder for
preparation of suspension for microbial enumeration. Ten mg of
each sample was weighed aseptically in sterile Eppendorf tubes and
sterile phosphate buffered saline was added to prepare serial dilutions
for counting colony forming units/mL Suspension was thoroughly
vortexed to get uniform suspension. Each dilution was spread inocu-
lated (0.1 mL) onto TSA and SDA plates and kept face up in biosafety
cabinet. TSA and SDA plates were incubated at 37C and 25C
respectively for 5 days. For detecting pathogenic bacteria E. coli and
Salmonella spp., samples were inoculated onto MacConkey’s Agar
and Xylose lysine decarboxylate agar respectively as per USP<62>
and incubated at 37C for 48h. Uninoculated plates were used as
negative control to ensure sterility of media.
Cell line culture
Madin-Darby Canine Kidney (MDCK) cells and African green mon-
key kidney epithelial Vero cells and Vero E6 cells were used in plaque
reduction assay. Cell lines were maintained in an incubator with 5%
CO2 at 37C. MDCK cells were grown in Minimum Essential Media
Alpha (MEM a) medium, with the addition of 5% heat-treated fetal
bovine serum (FBS, Sigma), 2 mM L-glutamine, 100 units/mL peni-
cillin, 1.25 mg/mL of amphotericin B (Fungizone), 50 mg/mL genta-
micin, 10 mM HEPES and 100 mg/mL streptomycin. Vero and
Vero E6 cells were grown in same medium with Dulbecco’s modified
Eagle’s medium in replacement of MEM a medium.
Plaque reduction assay
Bean gum powder was evaluated for their abilities to prevent infection
of Influenza virus strains H1N1 (A/California/7/2009-X181) and
H3N2 (A/Singapore/INFMH-16/0019/2016), Herpes Simplex virus
1 (KOS strain) and Herpes Simplex virus 2 (333 strain) using a quan-
titative viral plaque reduction assay.
Influenza virus
Prior to the assay, MDCK cells were plated in 48-well plates and incu-
bated for 24 h to reach 80–100% confluency. 330 mg Finely ground
bean gum powder was mixed with 1mL PBS, sonicated on ice to
release soluble FRIL protein and centrifuged. The supernatant was
www.moleculartherapy.orgcollected as sonicated bean gum extract. The sonicated bean gum
extract was diluted with PBS to reach various concentrations of
FRIL, and the Influenza strains was added to reach 1 pfu virus/mL
of Influenza. The samples were incubated at 4C on a rocker for 1
h, then added onto MDCK cells in 48-well plates for infection. The
plate was incubated with 5% CO2 at 37C for 1 h, then the virus mix-
tures were aspirated and washed to eliminate un-absorbed virus. The
cells were overlaid with serum-free MEM medium supplemented
with 0.3% Avicel, 0.3% Methylcellulose, 1 TPCK-treated trypsin,
and 0.1% BSA. The cells were incubated with 5% CO2 at 37C for
34 h, fixed and immune-stained with anti-Influenza nucleoprotein
antibody. Viral plaques were microscopically counted and analyzed
to generate dose-response curves.
Herpes simplex virus
Sonicated bean gum extract was dialyzed overnight to remove sugar
alcohols and other inferring compositions of bean gum. HSV virus
was co-incubated with increasing concentrations of sonicated bean
gum extract to reach 1 pfu virus/mL of HSV-1 or HSV-2. The samples
were incubated at 4C on a rocker for 1 h, then added onto Vero cells
in 48-well plates. Vero cells were then infected by adsorbing 100 mL of
the HSV FRIL mixtures at 34C for 45 min, then the virus mixtures
were aspirated and washed to eliminate un-absorbed virus. The cells
were overlaid with serum-free DMEM medium supplemented with
0.3% Methylcellulose. The cells were incubated with 5% CO2 at
37C for 50 h for HSV-1 and 40 h for HSV-2, fixed and stained in
4% formaldehyde and 0.2% crystal violet. Viral plaques were micro-
scopically counted and used to generate dose-response curves.Quantitation of vicine and convicine in lablab bean powder and
bean gum
Determination of vicine and convicine in faba beans by HPLC-DAD.
Vicine and convicine were determined based on the method
described by Gutierrez et al.102 Briefly, the samples were extracted
with water in a hot water bath for 3.5 h. Concentrated HCl was
then added to the sample extracts. Samples were analyzed by an Agi-
lent 1100 series high-performance liquid chromatography with a
diode array detector (HPLC-DAD). For the separation of com-
pounds, a C-18 -column was used with a gradient of phosphate buffer
and methanol.103 Vicine standard was purchased from Sigma -Al-
drich (St. Louis, MO, USA).Statistical analyses
Data of bean gum total dose quantification, release, ELISA, and pla-
que reduction assay was presented by means (standard deviation).
Statistical significance was calculated by One-Wau Electrochemical
impedimetric measures with an average of three duplicates.
GraphPad Prism 9.2 was used to plot and analyze graphs. The plaque
count was quantified manually using a dissecting microscope.DATA AND CODE AVAILABILITY
The raw data required to reproduce the above findings will be available from the corre-
sponding authors upon request.ACKNOWLEDGMENTS
Authors thank Dr. Che Ma (Genomics Research Center, Taiwan Academy of Sciences,
Taiwan) for providing lablab bean seed powder, purified FRIL and antibody, Prof.
Gary Cohen (University of Pennsylvania) and Roselyn J. Eisenberg (University of Penn-
sylvania) for HSV strains, antibody, Prof. Robert Ricciardi, Dr. Hancheng Guan (Univer-
sity of Pennsylvania) for different virus strains used in this study and guidance. The
authors thank Dr. Smruti Nair for help in conducting moisture content analysis. The au-
thors thank several collaborators at the University of Minnesota: Dr. Daniel Larranaga
Ordaz for help in operating the ART-5 mastication simulator, Drs. Alex Fok and Bonita
VanHeel for their help in modifying the ART-5 mastication simulator to include sound
human molars and related discussions. We also thank Riitta Henriksson (LUKE) for
HPLC sample preparation. Research performed in the Daniell lab was supported by
NIH grant R01 HL 107904.
AUTHOR CONTRIBUTIONS
HD conceived this project, designed experiments, interpreted data, participated in discus-
sions and wrote/edited several versions of this manuscript. YG performed assays on eval-
uation of purified FRIL, bean gum, and placebo gum potency against H1N1, H3N2,
HSV-1, and HSV-2, participated in discussions, created the graphic abstract, and wrote
the introduction section of the paper. UK performed H1N1, H3N2 plaque reduction as-
says using bean gum stored at different intervals of time and wrote results. RS participated
in determination of FRIL stability in bean powder and bean gum, and quantification of
FRIL release from the chewing in simulated chewing. RS performed HSV-1 and HSV-2
aggregation using ELISA assays, analyzed and wrote relevant sections. RS provided chew-
ing gum extract for plaque reduction assays. RJK performed release kinetic studies for
GFP and bean gums, analyzed and interpreted data, and wrote corresponding sections.
GW conducted bioburden and moisture content assessments for bean powder and
bean gum tablets as per USP guidelines. HK and J-MP performed vicine-convince anal-
ysis and interpreted data and wrote corresponding sections. GHC designed viral trap as-
says, provided HSV virus strains and developed antibodies required for these assays, re-
viewed several versions of this manuscript and helped in addressing questions from
reviewers. YG, RS, RJK, GW, and HK wrote results section of corresponding sections.
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.
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2024.
12.008.
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