nutrients
Article
Dietary Supplementation with Biobran/MGN-3 Increases
Innate Resistance and Reduces the Incidence of Influenza-like
Illnesses in Elderly Subjects: A Randomized, Double-Blind,
Placebo-Controlled Pilot Clinical Trial
Ahmed F. Elsaid 1, * , Sudhanshu Agrawal 2 , Anshu Agrawal 2






Citation: Elsaid, A.F.; Agrawal, S.;
Agrawal, A.; Ghoneum, M. Dietary
Supplementation with Biobran/
MGN-3 Increases Innate Resistance
and Reduces the Incidence of
Influenza-like Illnesses in Elderly
Subjects: A Randomized, Double-
Blind, Placebo-Controlled Pilot
Clinical Trial. Nutrients 2021, 13, 4133.
https://doi.org/10.3390/nu13114133
Academic Editor: Sathish Kumar
Natarajan
Received: 20 October 2021
Accepted: 14 November 2021
Published: 19 November 2021
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Attribution (CC BY) license (https://
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4.0/).
Nutrients 2021, 13, 4133. https://doi.org/10.3390/nu13114133
and Mamdooh Ghoneum 3
1 Department of Community Medicine and Public Health, Zagazig University, Zagazig 44519, Egypt
2 Division of Basic and Clinical Immunology, Department of Medicine, University of California,
Irvine, CA 92697, USA; sagrawal@uci.edu (S.A.); aagrawal@uci.edu (A.A.)
3 Department of Surgery, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059, USA;
mghoneum@ucla.edu
* Correspondence: elsaid_af@hotmail.com or afelsaid@zu.edu.eg
Abstract: Influenza-like illness (ILI) remains a major cause of severe mortality and morbidity in the
elderly. Aging is associated with a decreased ability to sense pathogens and mount effective innate
and adaptive immune responses, thus mandating the development of protective nutraceuticals.
Biobran/MGN-3, an arabinoxylan from rice bran, has potent anti-aging and immunomodulatory
effects, suggesting that it may be effective against ILI. The objective of the current study was to
investigate the effect of Biobran/MGN-3 on ILI incidence, natural killer (NK) cell activity, and the
expressions of RIG-1 (retinoic acid-inducible gene 1), MDA5 (melanoma differentiation-associated
protein 5), and their downstream signaling genes ISG-15 (interferon-stimulated genes 15) and MX1
(myxovirus (influenza) resistance 1, interferon-inducible). A double-blind, placebo-controlled clinical
trial included eighty healthy older adults over 55 years old, 40 males and 40 females, who received
either a placebo or Biobran/MGN-3 (500 mg/day) for 3 months during known ILI seasonality (peak
incidence) in Egypt. The incidence of ILI was confirmed clinically according to the WHO case
definition criteria. Hematological, hepatic, and renal parameters were assessed in all subjects, while
the activity of NK and NKT (natural killer T) cells was assessed in six randomly chosen subjects in
each group by the degranulation assay. The effect of Biobran/MGN-3 on RIG-1 and MDA5, as well
as downstream ISG15 and MX1, was assessed in BEAS-2B pulmonary epithelial cells using flow cy-
tometry. The incidence rate and incidence density of ILI in the Biobran/MGN-3 group were 5.0% and
0.57 cases per 1000 person-days, respectively, compared to 22.5% and 2.95 cases per 1000 person-days
in the placebo group. Furthermore, Biobran/MGN-3 ingestion significantly enhanced NK activity
compared to the basal levels and to the placebo group. In addition, Biobran/MGN-3 significantly up-
regulated the expression levels of RIG-1, MDA5, ISG15, and MX1 in the human pulmonary epithelial
BEAS-2B cell lines. No side effects were observed. Taken together, Biobran/MGN-3 supplemen-
tation enhanced the innate immune response of elderly subjects by upregulating the NK activity
associated with reduction of ILI incidence. It also upregulated the intracellular RIG-1, MDA5, ISG15,
and MX1 expression in pulmonary epithelial tissue cultures. Biobran/MGN-3 could be a novel
agent with prophylactic effects against a wide spectrum of respiratory viral infections that warrants
further investigation.
Keywords: Biobran/MGN-3; old adults; influenza-like illness; NK cell activity; RIG-1; MDA5; ISG-15;
MX1; degranulation assay; flow cytometry
1. Background
Influenza-like illness (ILI) is a viral infection with characteristic annual seasonality
that leads to around 3–5 million severe illnesses and an estimated 500,000 annual deaths
https://www.mdpi.com/journal/nutrients
Nutrients 2021, 13, 4133 2 of 14

around the world [1,2]. The World Health Organization (WHO) has recently revised the
definition of ILI to be “acute respiratory illness with a measured temperature of ≥38 ◦ C
and cough, with onset within the past 10 days” [3]. The diagnosis of influenza itself
cannot be made without virological confirmation, which is costly and rarely performed
in public health surveillance systems. To enhance the sensitivity of influenza surveillance
systems to capture influenza, ILI has been recommended by the WHO to be used as a
surrogate for influenza infection [4]. The simple clinical criteria used to define ILI was
associated with low specificity. Therefore, in most surveillance systems around the world,
a wide array of viruses has been isolated from ILI cases, including the respiratory syncytial
virus, metapneumovirus, corona viruses, adenoviruses, rhinoviruses, and others [4–7]. In
Egypt, during the period from 2012 to 2015, influenza viruses were isolated from only
13% of ILI cases identified in 13 different sentinel surveillance sites [8]. ILI infection and
complications are particularly dangerous in the elderly population, due to the phenomenon
of immunosenescence or age-associated decline of immune system activity [9,10].
In order for the body to mount a rapid antiviral immune response, it must detect
intracellular viral pathogens. RIG-I-like receptors (RLRs), a family of pattern-recognition
receptors (DExD/H box RNA helicases), play a key role in the induction of the innate
immune response by sensing viral nucleic acid [11]. RLRs are expressed in the cytoplasm of
almost all mammalian cells and include RIG-I (retinoic acid-inducible gene 1, also known
as DDX58), MDA5 (melanoma differentiation-associated protein 5, also known as IFIH1),
and LGP2 (laboratory of genetics and physiology, DHX58) receptors. Current evidence
suggests that RIG-1 and MDA5 can recognize different nucleic acids patterns and capture
different byproducts of viral nucleic acid replication and metabolism. RIG-1 has been
reported to recognize 50 phosphorylated short double-stranded RNA (50 ppp-dsRNA) and
the U/A-rich 30 regions of viral RNA, while MDA5 has been reported to recognize long
dsRNA [12,13]. This differential nucleic acid-sensing capacity allows RIG-1 and MAD5
to detect a wide spectrum of infectious viruses. Activation of RIG-1 and MAD5 leads to
activation of interferon-I α/β (IFN-I α/β) with their downstream gene network, collectively
referred to as interferon-stimulated genes (ISG), such as ISG15 and MX1 [14–16]. Both
ISG15 and MX1 have been shown to protect against several viruses including influenza
infection [17–19].
Another essential component of the innate immune response is NK cells, a type of
effector cytotoxic lymphocyte. NK cells can engage and lyse cells expressing foreign
antigens such as virally-infected and tumor cells [20–24]. The pivotal role of NK cells
in controlling various viral infections has been demonstrated for the cytomegalovirus
(CMV) [25], hepatitis C virus infection [26], HIV infection [27,28], and viral infection of the
lungs [29]. Some of the mechanisms implicated in NK cell-mediated target cell lysis include
the release of perforin and granzymes into target cells, the activation of death receptors
and induction of apoptosis in target cells, and the release of several pro-inflammatory
cytokines [30,31]. It is well established that aging is associated with a decline in NK cell
activity concomitant with a reduction of perforin and granzyme expression, which has
been proposed as a plausible mechanism for the increased susceptibility of the geriatric
population to viral infections and carcinogens [32–34].
The natural arabinoxylan rice bran product called Biobran/MGN-3 has been studied as
an immunomodulator as well as a beneficial product for aged individuals. Biobran/MGN-3
has been extensively studied for its ability to enhance NK cell activity in both experimen-
tal animals and humans [35–38]. Interestingly, Biobran/MGN-3 has also been shown
to counteract age-associated decline of NK cell activity in aged mice [39] and in the
geriatric population [40]. Furthermore, Biobran/MGN-3 has been reported to induce
potent immunomodulatory effects. For example, in vitro studies of human monocyte-
derived dendritic cells, phagocytic cells, and peripheral blood lymphocytes have shown
that Biobran/MGN-3 can modulate the production of cytokines such as tumor necro-
sis factor-α (TNF-α), interferon-gamma (IFN-γ) and -lambda (IFN-λ), and interleukin-2
(IL-2) and IL-12 [36,37,41–43]. The potential use of Biobran/MGN-3 against various types
Nutrients 2021, 13, 4133 3 of 14

of viral infections such as HIV and HCV has also been previously investigated [44–46],
and Biobran/MGN-3 has been shown to exhibit a psychoneuroimmune modulatory ef-
fect by enhancing health-related quality of life in healthy older adults [47] and in cancer
patients [48,49].
The well-documented immunomodulatory effect of Biobran/MGN-3 and its capacity
to restore the age-associated decline of NK cell activity prompted us to investigate its pro-
phylactic efficacy against virus-induced ILI. To gain insight into the involved mechanisms,
we assessed NK cell activity in the studied subjects as well as the expression levels of
intracellular RIG-1, MDA5, and the downstream genes ISG15 and MX1 in lung epithelial
BEAS-2B cells.

2. Subjects and Methods

2.1. Trial Design
A double-blind placebo-controlled design was utilized in the current clinical trial
to assess the effect of Biobran/MGN-3 on the incidence of ILI in older adults. Figure 1
depicts a simple flow diagram of the clinical trial. The study spanned the time period from
November 2018 until the end of February 2019, a period with known peak incidence of ILI
attacks in Egypt [8]. The study protocol conformed to the ethical guidelines of the 1975
Declaration of Helsinki and was approved by the Institutional Review Board, Zagazig
University Hospital, Faculty of Medicine (IRB# 1507/1/6/2017). The clinical trial was
registered at clinicaltrials.gov with the ID# NCT04646980 on 30 November 2020.

2.2. Participants
A total of 130 subjects ≥ 56 years was initially recruited from the visitors of outpatient
clinics at Zagazig University Hospitals, Egypt, and assessed for eligibility to participate in
the clinical trial. Only local residents of Zagazig city were recruited in order to facilitate
follow-up home visits. Thirty-seven subjects (n = 37, 19 ♀, 18 ♂) did not meet the inclusion
criteria and 10 subjects refused to participate when they were informed that the intervention
included unlabeled sachets. Three randomly chosen males by a statistician ignorant to
the study were not enrolled in the study to adjust the males/females ratio to 1. Finally,
eighty apparently healthy older adults ≥ 56 years, 40 males and 40 females, were enrolled
in the study. Subjects ≥ 56 years were recruited to the study in order to meet the WHO
definition of old age in African nations [50]. In addition, this age is close to public service
retirement in Egyptian society, which is associated with significant social, mental, and
psychological stress and therefore could be associated with significant decline in NK cell
activity. Close monitoring, daily follow-up of subjects, and the relatively short period of
the study (3 months) resulted in the absence of any dropouts.

2.3. Inclusion and Exclusion Criteria

Inclusion criteria included older adults ≥ 56 years who were local residents of Zagazig,
Egypt, at the time of the study and who were willing to participate voluntarily in the
study. Local residents were chosen to ensure close monitoring and to reduce dropout rate.
Exclusion criteria included subjects who had taken an influenza vaccine, cortisone, or any
other immunosuppressive agents such as radiation or chemotherapy at the time of the
study. Exclusion criteria also included subjects with a history of chronic infections (e.g.,
TB), malignancies, auto-immune disorders, liver or kidney failure, and major psychological
insults for probable un-cooperation with instruction.

2.4. Consent
Informed consents were obtained from all participants after explaining the study
purpose and design.
Nutrients 2021, 13, 4133 4 of 14

Assessed for eligibility (n = 130)

Enrollment

Excluded (n = 50)
♦ Not meeting inclusion criteria (n = 37, 13 ♂ ,14 ♀)
╪
♦ Declined to participate (n = 10, 4 ♂ + 6 ♀ )
¥
♦ Other reasons (n = 3)

§
Randomized (n = 80, 40 ♂ + 40 ♀ )

Allocation

♦ Biobran/MGN-3 (n = 40, 20 ♂ + 20 ♀ ) ♦ Placebo (n = 40, 20 ♂ + 20 ♀ )

♦ Received placebo 500 mg/day
♦ Received Biobran/MGN-3 500 mg/day

Follow-Up
Nutrients 2021, 13, x FOR PEER REVIEW

Ø Ø
♦ Lost to follow-up (n = 0) ♦ Lost to follow-up (n = 0)

♦ Discontinued intervention (n = 0) ♦ Discontinued intervention (n = 0)

ALP (U/L) 185.5 (51.3)
ALT (U/L) 22.0 (9.3)
AST (U/L) 17.7 (4.6)
Analysis UA (mg/dL) 7.4 (2.9)
RBCs (×10 /μL)
6 4.8 (0.5)
♦ Analysed (n = 40, 20 ♂ + 20 ♀ ) ♦ Analysed (n = 40, 20HB
♂ + (g/dL)
20 ♀ ) 12.1 (1.7)
♦ Excluded from analysis (n = 0) HCT
♦ Excluded from analysis %
(n = 0) 37.4 (4.4)
MCHC (g/dL) 32.3 (0.9)
WBC (×103/μL) 6.8 (1.8)
Figure 1. Simple flow diagram showing different stages of the clinical trial. ǂ Declined
Biobran/MGN-3 to participate%when informed
Neutrophils that
57.3 (14.3)
¥ (500 mg/day)
the intervention included unlabeled coded sachets. Not enrolled to adjust males/females ratio to 1. § The principal
Lymphocytes % 32.5 (7.9)
for 3procedure,
investigator was not involved in the randomization or allocation months which was performed by a statistician ignorant
Platelets (×10 3/μL) 187.3 (37.1)
of the study. Ø Close monitoring, daily follow-up of subjects, and the relatively short period of the study (3 months) resulted
ALP (U/L) 213.0 (43.0)
in the absence of any dropouts.
ALT (U/L) 22.0 (7.2)
2.5. Randomization and Allocation Concealment AST (U/L) 23.8 (11.0)
UA (mg/dL)
Males (n = 40) and females (n = 40) were randomly allocated into the two study 5.9 (2.5)
arms
§ p-values represent
at a ratio of 1:1 to yield 40 subjects/arm comparison
(20 males between post-treatment
and 20 females). Randomizationand and
baseline valu
because all data were normally distributed. All comparisons yielded non-si
allocation were performed by a statistician ignorant of the study using SPSS software.
ment and the basal levels. ǂ Comparison between placebo and Biobran/MGN
2.6. Intervention with regards to all variables. p ≤ 0.05 was considered statistically significant.
hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemo
Biobran/MGN-3 or placebo, 500 mg/day
aminotransferase; AST,for 3 months,
aspartate was used inUA,
aminotransferase; theuric
form
acid.of
powder enclosed in sachets. Biobran/MGN-3 is a denatured hemicellulose whose main
chemical structure is an arabinoxylan with a xylose 3.2.in its main chain and
Biobran/MGN-3 an arabinose Significa
Supplementation
polymer in its side chain. It is obtained by reacting rice bran hemicellulose with several
Figure 2 shows the incident cases and
incidence rate in the Biobran/MGN-3 group
than the incidence rate in the placebo group
two-tailed Fisher’s exact test). The risk of IL
estimated to be 18.2% (CI = 3.9 − 48.9), which
43.4 − 66.3) value for the placebo group (p
Nutrients 2021, 13, 4133 5 of 14

carbohydrate-hydrolyzing enzymes from Shiitake mushrooms, yielding a polysaccharide

that contains β-1,3-glucans and activated hemicelluloses [44].

2.7. Ingredients of Biobran/MGN-3 Sachets

The Biobran/MGN-3 sachet’s ingredients included Biobran: 500 mg, hydroxypropyl
distarch phosphate: 280 mg, dextrin: 200 mg, tricalcium phosphate: 20 mg, and maltitol:
1000 mg. The placebo sachet’s ingredients included maltitol: 1000 mg, hydroxypropyl
distarch phosphate: 780 mg, dextrin: 200 mg, and tricalcium phosphate: 20 mg.

2.8. Blinding
To ensure blinding, sachets of Biobran/MGN-3 and placebo, kindly provided by
Daiwa Pharmaceuticals Co., Ltd., Tokyo, Japan, were indistinguishable except by imprinted
letter code. The manufactured company provided the Biobran/MGN-3 and placebo pow-
der in a formulation that could not be differentiated by the shape, smell, color, or even
taste. Both participants and health care provider were blinded to the intervention during
all phases of the study. The code was only revealed after complete analysis of the results.

2.9. Follow-Up
Participants were instructed to call upon the health care giver upon the incidence of
any complaint, ailment, fever, cough, or diarrhea. In addition to daily follow-up by phone,
participants were offered weekly home visits to closely monitor their health status. No vi-
tamins or any over-the-counter drugs were allowed during the study without consultation.
ILI diagnosis was documented by the incidence of acute respiratory illness, cough with
temperature ≥ 38 ◦ C [3]. Diagnosed ILI cases received the proper treatment by the health
care giver.

2.10. Outcome
The primary outcomes of the current trial included (i) the incidence of ILI infection in
the Biobran/MGN-3 arm compared to the placebo arm and (ii) confirmation of the effect of
Biobran/MGN-3 on NK cell activity. ILI diagnosis was made by documenting the incidence
of acute respiratory illness, cough and temperature of ≥38 ◦ C [3]. The incidence rate was
calculated by dividing the number of incident ILI cases by the number of participants in
each group. The incidence density was calculated by dividing the number of incident cases
by the total person-time at risk [51].
The secondary outcome included the incidence of any clinically reported or laboratory-
observed adverse effect.

2.11. Sample Size

To our knowledge, the prophylactic effect of Biobran/MGN-3 on reducing the in-
cidence of ILI was not previously studied. As such, the current pilot clinical trial was
conducted with 80 subjects to investigate the efficacy of Biobran/MGN-3 in reducing ILI
incidence. The effect of Biobran/MGN-3 on NK cell activity was measured in six randomly
selected subjects from each group because this number was shown to be sufficient for
detecting a significant difference between Biobran/MGN-3 and the placebo [40].

2.12. Laboratory Investigations

Blood samples (5 cc) were collected from a randomly selected six subjects in each
group for laboratory investigation. Alanine aminotransferase (ALT/SGPT) and aspartate
aminotransferase (AST/SGOT) were used to monitor liver functions, and serum uric acid
was used to monitor kidney function. The following hematological parameters, red blood
cell count (RBC), total hemoglobin (Hb), mean corpuscular hemoglobin (MCH), hematocrit
(HCT), mean corpuscular volume (MCV), and total and differential white blood cells count,
were also assessed.
Nutrients 2021, 13, 4133 6 of 14

2.13. Degranulation Assay for NK Cell Activity

The activity of NK cells was measured using the degranulation assay as previ-
ously described by Elsaid et al. [40]. Heparinated blood (100 µL) was collected to ex-
amine NK activity under 4 conditions: (1) unstimulated condition (RPMI 1640 medium
alone); (2) stimulated condition (combination of phorbol-12-myristate-13-acetate (PMA,
50 ng/mL, Sigma, St. Louis, MO, USA) and Ca2+ ionophore (ionomycin, 250 ng/mL,
Sigma, St. Louis, MO, USA); (3) IgG isotypic staining negative control; and (4) positive
control (PMA/ionomycin plus cytochalasin (5 µg/mL, Sigma, St. Louis, MO, USA). For
staining, samples were incubated with FITC-labeled mouse anti-human CD-107a (clone
H4A3, BD Bioscience, San Jose, CA, USA) for 5 h at 37 ◦ C under 5% (v/v) CO2 . One hour
later, monensin (GolgiStop, BD Bioscience, San Jose, CA, USA) was added to a final con-
centration of 6 µg/mL. To stain the IgG isotypic negative control, samples were incubated
with mouse anti-human FITC-labeled IgG AB (clone G18-145, BD Bioscience, San Jose, CA,
USA) [52]. At the end of incubation, RBCs were lysed using BD PharmLyse (BD Bioscience).
Cells were spun down by centrifugation (300× g, 5 min), washed once, and re-suspended
with 5 µL of PE-labeled mouse anti-human CD56 AB clone R19-760 (NCAM-1, BD Bio-
science) and 5 µL of PerCP-labeled mouse anti-human CD3 clone SK7 (BD Bioscience)
in a final volume of 100 µL staining buffer for 15 min at 37 ◦ C. Cells were collected with
centrifugation (300× g, 5 min), washed once, and re-suspended in 100 µL FACS buffer.
BD FACS Calibur with CellQuest software was used for analysis (BD Bioscience, San Jose,
CA, USA).

2.14. Cell Culture and Flow Cytometry

BEAS-2B cells (ATCC) were grown in bronchial epithelial growth medium using the
BEGM Bullet kit (Lonza, Walkersville, MD, USA) on collagen-coated (5 g/cm2 ) plates
and maintained at 37 ◦ C, 5% CO2 . Cells were then seeded in 6-well plates and grown
until 90% confluent, then incubated with either Biobran/MGN-3, 100 µg/mL (Daiwa
Technologies, Japan), for 72 h or PBS (control). After harvesting, cells were washed with
PBS, permeablized and fixed using BD cytofix/cytoperm buffer as recommended by the
manufacturer. Cells were then spun down, washed, and re-suspended in 100 µL of the
first block solution with 10% rabbit serum for 30 min at room temperature. Staining was
performed by incubating the cells for 15 min at 37 ◦ C with 5 µL of either fluorescent-
labeled monoclonal anti-RIG-1 PE (catalog # sc-376845 PE, Santa Cruz Biotechnology,
Dallas, TX, USA), monoclonal rabbit anti-human MX1 AB (catalogue # orb228747, Biorybt,
San Francisco, CA, USA), monoclonal rabbit anti-human MDA5 AB, clone # 33H12L34
(catalog # 700360, ThermoFisher Scientific, Rockford, IL, USA), monoclonal rabbit anti-
human anti-ISG15 AB (catalog # ab133346, Abcam, Cambridge, MA, USA), or the IgG
isotype-matched control AB (negative control). For RIG-1, cells were washed once in FACS
buffer, collected by centrifugation, re-suspended in FACS buffer, and analyzed. For 2 ry
AB staining of MDA5, MX1, and ISG15, cells were washed in permeablized/fixed BD
cytofix/cytoperm buffer, collected by centrifugation, then re-suspended in staining buffer
with 10% goat serum (secondary block) for 30 min at room temperature. Finally, cells were
incubated with PE-conjugated goat anti-rabbit IgG secondary antibody (catalog # F0110,
R&D Systems, Minneapolis, MN, USA) at 10 µL/106 cells. Data acquisition and analysis
were performed using FACS Calibur (BD Biosciences) and BD Cell Quest software (BD
Biosciences). All cell culture and flow experiments were performed in triplicate.

2.15. Statistical Analysis

Continuous variables were presented as mean ± standard deviation (SD). Data nor-
mality was checked using the Shapiro–Wilk test. Comparison of quantitative variables
between groups was performed using a two-tailed Student’s t-test, whereas comparison
within groups (post-intervention vs. basal levels) was performed using paired t-test. Com-
parison of the incidence rates and density between Biobran/MGN-3 and placebo groups
was performed using the Fisher’s exact test. Level of significance of all statistical tests was
 Nutrients 2021, 13, 4133 7 of 14

set at an alpha level of significance ≤ 0.05. OpenEpi, version 3.01, and SPSS, version 20,
were used for statistical analyses [53,54].

3. Results
3.1. Biobran/MGN-3 Did Not Adversely Affect the Hematological, Liver, and Kidney Functions
The average age of participants in the Biobran/MGN-3 group (61.5 ± 5.6 years) was
nearly equal to that in the placebo group (62.6 years ± 5.7). Results depicted in Table 1
show that all basal level parameters of hematological, liver, and kidney functions were
not significantly different between the two groups. This indicates that Biobran/MGN-3
did not adversely affect any of the hematological, liver, or renal functions. This finding is
consistent with previous studies demonstrating that Biobran/MGN-3 is safe without any
Nutrients 2021, 13, x FOR PEER REVIEW 8 of 14
adverse effects [46,49,55,56].

Table 1. Comparison of hematological indices and liver and kidney functions within groups (before and after treatment)
and between groups (Biobran/MGN-3
ALP (U/L) and placebo groups).
185.5 (51.3) 183.2 (50.1) 0.66
Treatment ALT (U/L) Parameter 22.0 (9.3) Baseline 20.3 (8.4)Post-Treatment 0.15 p-Value §
AST (U/L)
RBCs (×106 /µL)
17.7 (4.6) 4.3 (0.5) 18.3 (5.8) 4.1 (0.5) 0.07 0.58
UA (mg/dL) 7.4 (2.9) 8.2 (3.8) 0.51
HB (g/dL) 11.3 (1.5) 13.8 (1.4) 0.40
RBCs (×106/μL) 4.8 (0.5) 4.7 (0.6) 0.18
HCT % 35.7 (4.0) 36.7 (3.5) 0.51
HB (g/dL) 12.1 (1.7) 13.9 (1.6) 0.93
HCT MCHC % (g/dL) 37.4 (4.4) 27.3 (2.7) 36.9 (3.1) 37.6 (0.7) 0.6 0.80
3
(×10 /µL)
MCHC WBC (g/dL) 32.3 (0.9) 5.7 (0.9) 37.4 (1.5) 6.1 (1.2) 0.46 0.76
WBC (×10Neutrophils
3 /μL) % 6.8 (1.8) 56.1 (14.5) 6.7 (1.4) 57.3 (11.6) 0.68 0.32
Placebo ǂ
Biobran/MGN-3
Neutrophils %
Lymphocytes % 57.3 (14.3) 33.3 (10.2) 60.7 (7.3) 39.5 (9.8) 0.37 0.23
(500 mg/day)
Nutrients 2021, 13, x FOR PEER REVIEW 8 of 14
Lymphocytes %
Platelets (×10 /µL) 3 32.5 (7.9) 218.8 (48.4) 38.8 (8.5) 231.5 (48.0) 0.24 0.19
for 3 months
Platelets (×103/μL) 187.3 (37.1) 208.8 (16.9) 0.12
ALP (U/L) 185.5 (51.3) 183.2 (50.1) 0.66
ALP (U/L) 213.0 (43.0) 195.2 (35.1) 0.58
ALT (U/L) 22.0 (9.3) 20.3 (8.4) 0.15
ALTALP (U/L) (U/L) 22.0185.5
(7.2)(51.3) 19.3183.2
(6.6)(50.1) 0.38 0.66
ASTALT (U/L) (U/L)
AST (U/L) 22.0 (9.3)
23.8 (11.0) 17.7 (4.6) 20.2 20.3
(8.8)(8.4)
18.3 (5.8) 0.26 0.150.07
AST
UA (mg/dL) UA (U/L)
(mg/dL) 17.7
5.9 (2.5) (4.6)7.4 (2.9) 18.3
6.7 (1.8) (5.8)
8.2 (3.8) 0.33 0.070.51
§ p-values represent comparison UA (mg/dL)
between
RBCs 106 /µL)
(×post-treatment 7.4baseline
and (2.9)4.8 (0.5)
values and were 8.2 (3.8)4.7 (0.6) using paired0.51
determined t-test
0.18
because all data were normally RBCs (×106/μL)
distributed. All comparisons4.8 (0.5) non-significant differences
yielded 4.7 (0.6) between the post-treat- 0.18
HB (g/dL) 12.1 (1.7) 13.9 (1.6) 0.93
ment and the basal levels. ǂ Comparison HB (g/dL) between placebo and Biobran/MGN-3
12.1 (1.7) groups13.9
yielded
(1.6)non-significant differences 0.93
with regards to all variables. p ≤ 0.05 wasHCT % 37.4 (4.4) RBC, red blood36.9 cell;(3.1) 0.6
HCT %considered statistically significant.
37.4 (4.4) 36.9 (3.1)
Nutrients Hb, hemoglobin;
2021, HCT,
0.6
13, x FOR PEER REVIEW
hematocrit; MCV, mean corpuscularMCHC volume; MCH, mean corpuscular
(g/dL) 32.3hemoglobin;
(0.9) WBC, white blood cell; ALT, alanine
37.4 (1.5) 0.46
MCHC (g/dL) 32.3 (0.9) 37.4 (1.5) 0.46
aminotransferase; AST, aspartate aminotransferase; UA, uric acid.
WBC WBC (×10(× 103 /µL)
3/μL) 6.8 (1.8)6.8 (1.8) 6.7 (1.4)6.7 (1.4) 0.680.68
Biobran/MGN-3 ǂ
Biobran/MGN-3
(500mg/day)
mg/day) 3.2.Neutrophils
Neutrophils
Biobran/MGN-3 % %Supplementation 57.3 (14.3)
57.3 (14.3)
Significantly 60.7 (7.3)
Reduced the60.7
ILI(7.3)
Incidence Rate and 0.37Density
0.37 ALP (U/L)
(500
for 3 months Lymphocytes
Lymphocytes
Figure 2 shows% %the incident 32.5 (7.9)
cases (7.9) the day38.8
32.5 and (8.5)
of ILI 38.8
onset(8.5)in the two groups. 0.240.24TheALT (U/L)
for 3 months
Platelets
incidence
Platelets(×10 3
rate(×in /μL)3
10the 187.3 (37.1)
Biobran/MGN-3
/µL) group
187.3 (37.1)was 5%208.8 (16.9)
(2/40), which
208.8 was significantly
(16.9) 0.120.12
lowerAST (U/L)
than ALP (U/L)
the incidence
ALP (U/L) rate in the213.0 (43.0)
placebo group
213.0 (43.0) which 195.2
reached(35.1)22.5%
195.2 (35.1) (9/40) (p= 0.58
0.048 0.58 UA
using a (mg/dL)
two-tailed
ALT (U/L) Fisher’s exact test).22.0 The(7.2)
risk of ILI infection 19.3in(6.6)
the Biobran/MGN-3 0.38 group was RBCs (×106/μL)
ALT (U/L) 22.0 (7.2) 19.3 (6.6) 0.38
estimated
AST (U/L) to be 18.2% (CI = 3.9 − 48.9),
23.8 (11.0)which was significantly
20.2 (8.8) lower than the 55.1% 0.26 (CI =HB (g/dL)
43.4UA − 66.3)ASTvalue(U/L)for the placebo group 23.8 (11.0)
(p < 0.05). The 20.2ratio
risk (8.8) of ILI infection0.26
(mg/dL) 5.9 (2.5) 6.7 (1.8) 0.33 in the HCT %
Biobran/MGN-3UA (mg/dL) group compared to the
5.9 placebo
(2.5) group
§ p-values represent comparison between post-treatment and baseline values and were determined using paired was estimated
6.7 (1.8) to be 0.33, using
0.33 the
t-test
MCHC (g/dL)
because
§ p-valuesall data were
represent Taylor
normally
comparison series approximation.
distributed.
between The
All comparisons
post-treatment estimated
yielded
and baseline values preventive
andnon-significant fraction
were determineddifferences in the
using pairedbetween Biobran/MGN-3
the all
t-test because post-treat-
data were
WBC (×103/μL)
ment anddistributed.
normally the basal levels. ǂ Comparison
Allgroup was almost
comparisons yielded 82% (95%
between CIdifferences
placebo
non-significant =and
9.9% − between
96.4%).the
Biobran/MGN-3 Similarly,
groups significant
yielded
post-treatment results
Biobran/MGN-3
andnon-significant
the basal were
levels. ǂdifferences
obtained
Comparison
between placebo andvariables.
Biobran/MGN-3 groups yielded non-significant differences p ≤hemoglobin; Neutrophils %
with regards to all when pcomparing
≤ 0.05 was considered
the incidencestatistically
density rates inwith
significant. regards
theRBC,
twored toblood
all variables.
groups. cell; incidence
The
(500 Hb,
mg/day) 0.05 was considered
density HCT,
rate
statistically significant. RBC,
hematocrit; MCV, meanincorpuscularred blood
thecell;
cell; Hb, hemoglobin;
volume; MCH,
Biobran/MGN-3
HCT,
mean
group
hematocrit;
corpuscular
was estimated
MCV, mean
hemoglobin; corpuscular
be 0.57 WBC,
volume;
white MCH,
blood mean Lymphocytes %
corpuscular
cell; ALT, alanine
hemoglobin; WBC, white blood ALT, alanine aminotransferase; AST, aspartateto cases UA,
aminotransferase; perfor1000
uric person-days
3 acid.
months (95%
aminotransferase; AST,CI aspartate aminotransferase; UA, uric acid.
= 0.06 − 2.06) compared to 2.95 cases per 1000 person-days (95% CI = 1.34 − 5.59) in the Platelets (×103/μL)
placebo group (p = 0.038 using a two-tailed Fisher’s exact test). The risk ratio of ILI infec-ALP (U/L)
tion 3.2.
in theBiobran/MGN-3
Biobran/MGN-3 Supplementation
group compared Significantly Reduced
to the placebo the ILI
group was Incidence
estimated Ratetoand Density
be 0.19 ALT (U/L)
Figure 2 shows the incident cases and the day
using the Byar method. The preventive fraction in the Biobran/MGN-3 group was esti-AST of ILI onset in the two groups. The (U/L)
incidence rate in the
mated to be 81% (95% CI = 10.5% − 95.8%).Biobran/MGN-3 group was 5% (2/40), which was significantly lower
UA (mg/dL)
than the incidence rate in the placebo group which reached§ 22.5% p-values (9/40) (p= 0.048
represent using a between p
comparison
two-tailed Fisher’s exact test). The risk of ILI infection in the Biobran/MGN-3
because all data weregroup normally was distributed.
estimated to be 18.2% (CI = 3.9 − 48.9), which was significantly mentlower
and the than
basalthelevels.
55.1% ǂ Comparison
(CI = betw
Nutrients 2021, 13, 4133 8 of 14

3.2. Biobran/MGN-3 Supplementation Significantly Reduced the ILI Incidence Rate and Density
Figure 2 shows the incident cases and the day of ILI onset in the two groups. The
incidence rate in the Biobran/MGN-3 group was 5% (2/40), which was significantly lower
than the incidence rate in the placebo group which reached 22.5% (9/40) (p = 0.048 using
a two-tailed Fisher’s exact test). The risk of ILI infection in the Biobran/MGN-3 group
was estimated to be 18.2% (CI = 3.9 − 48.9), which was significantly lower than the 55.1%
(CI = 43.4 − 66.3) value for the placebo group (p < 0.05). The risk ratio of ILI infection in
the Biobran/MGN-3 group compared to the placebo group was estimated to be 0.33, using
the Taylor series approximation. The estimated preventive fraction in the Biobran/MGN-3
group was almost 82% (95% CI = 9.9% − 96.4%). Similarly, significant results were obtained
when comparing the incidence density rates in the two groups. The incidence density
rate in the Biobran/MGN-3 group was estimated to be 0.57 cases per 1000 person-days
(95% CI = 0.06 − 2.06) compared to 2.95 cases per 1000 person-days (95% CI = 1.34 − 5.59)
in the placebo group (p = 0.038 using a two-tailed Fisher’s exact test). The risk ratio of ILI
infection in the Biobran/MGN-3 group compared to the placebo group was estimated to
Nutrients 2021, 13, x FOR PEER REVIEW 9 of 14
be 0.19 using the Byar method. The preventive fraction in the Biobran/MGN-3 group was
estimated to be 81% (95% CI = 10.5% − 95.8%).

Figure2.2.ILI
Figure ILIincidence
incidencedensity
densitydistribution
distribution among
among the
the MGN-3/Biobranand
MGN-3/Biobran andplacebo
placebogroups.
groups.The
The x-
axis represents the patient number and y-axis represents the day of onset of
x-axis represents the patient number and y-axis represents the day of onset of ILI.ILI.

3.3.
3.3.Biobran/MGN-3
Biobran/MGN-3Specifically Enhanced
Specifically EnhancedNKNKCell Activity
Cell butbut
Activity NotNotNKT Activity
NKT Activity
Supplementation with Biobran/MGN-3 at 500 mg/day significantly
Supplementation with Biobran/MGN-3 at 500 mg/day significantly enhanced enhanced NK NK
cell cell
activity,
activity,asassuggested
suggestedbyby
the percentage
the percentageof of
CD107a-expressing
CD107a-expressing NKNK cellscells
uponuponstimulation
stimulation
with
withPMA/ionomycin
PMA/ionomycincomparedcomparedtotobasal
basallevels asas
levels well
wellas as
to to
thethe
placebo
placebo group
group(Table 2). 2).
(Table
This effect of Biobran/MGN-3 was specific to NK cells; we did not observe a similar
This effect of Biobran/MGN-3 was specific to NK cells; we did not observe a similar effect effect
ininNKT
NKTcells.
cells.

Table 2. Comparison of NK, NKT, and CD-107a-expressing NK and NKT cells within groups (before and after treatment)
and between groups (Biobran/MGN-3 and placebo groups).

Treatment Parameter Baseline Post-Treatment p-Value §

NK 5.3 (1.9) ǂ 6.0 (1.7) ǂ 0.62
NKT 4.5 (1.6) ǂ 5.6 (2.4) ǂ 0.44
Placebo
NK CD-107 a 45.3 (12.0) ǂ 50.8 (19.5) ¥ 0.38
 Nutrients 2021, 13, x FOR PEER
Nutrients
REVIEW
2021, 13, x FOR PEER REVIEW
Nutrients 2021, 13, x FOR PEER REVIEW
Nutrients 2021, 13, x FOR PEER
Nutrients ALP
REVIEW
2021, 13,(U/L) ALP
x FOR PEER REVIEW185.5
(U/L)
(51.3) 18
ALP ALT (U/L) ALT22.0
(U/L)
(9.3) 2
Nutrients 2021, 13, x FOR PEER
Nutrients
REVIEW
2021, 13, x(U/L) ALP
FOR PEER REVIEW 185.5
(U/L)
(51.3) 18
Nutrients 2021, 13, 4133 AST (U/L) AST17.7
(U/L) 9 of 14
(4.6)
ALT (U/L) ALT22.0 (9.3) 21
Nutrients 2021, 13, x FOR PEER
Nutrients 2021,ALP
REVIEW 13, x FOR PEER REVIEW 185.5 (51.3)
UA AST (mg/dL)
(U/L) UAAST (mg/dL)
7.4
17.7
(U/L) (2.9)
(4.6)
ALTALP
Nutrients 2021, 13, x FOR PEER REVIEW
(U/L) ALP 22.0
185.5 (U/L) (9.3)
(51.3) 11
RBCs
UA AST (×10
(mg/dL) 6/μL)
(U/L) RBCsUA (×104.8
(mg/dL)
7.4
17.7
6(0.5)
/μL)
(2.9)(4.6)
ALP ALT (U/L) ALT
ALP 185.5 22.0
(U/L)
(U/L) (9.3)
(51.3) 18
Table 2. Comparison of NK, NKT, and CD-107a-expressing NutrientsNK 2021, 13,NKT
and x FORcells
Nutrients
PEER REVIEW
within 2021, HB 13,
groups x FOR
(g/dL) (before PEER andREVIEW
afterHB (g/dL)
12.1
treatment) (1.7) 1
RBCs UA AST (×10
(mg/dL) 6/μL)
(U/L) RBCs AST (×104.8
7.4
17.7
6(0.5)
(U/L) /μL)
(2.9)
(4.6)
and between groups (Biobran/MGN-3 and placebo groups).
ALT
ALP (U/L) ALT
ALP 22.0
185.5 (U/L) (9.3)
(51.3) 18 2
RBCs HCT
HB (g/dL) (×10 % 6/μL) HCT
HB (g/dL)37.4
12.14.8% (4.4)
(1.7)
(0.5) 13
UA
AST
ALT (mg/dL)
(U/L) UA
AST
ALT185.5 (mg/dL)
17.77.4
(U/L)
22.0 (2.9)
(4.6)
(9.3) 231
MCHC ALP
HCT (U/L)
(g/dL)
%6/μL) MCHC HCT 32.3
37.4 (g/dL)
%(0.9) (51.3)
(4.4)
Treatment Parameter Baseline RBCs
UA HB
Post-Treatment(g/dL)
(×10
(mg/dL) RBCs
UA p-Value 12.1
(mg/dL)4.8
(×10
7.4 (2.9)(1.7)
6(0.5)
/μL)
§
WBC AST
ALT
ALP (×10(U/L)
(U/L)
3/μL) WBC AST
ALP 17.7
(U/L)
22.0
185.5
(×10 (U/L)
6.8 (4.6)
3(1.8)
/μL)(9.3)
(51.3) 131
Nutrients 2021, 13, x FOR PEER REVIEW MCHC HCT (g/dL)
6%
MCHC 32.3 (g/dL)
37.4 (0.9)
(4.4)
NK Biobran/MGN-3 5.3 (1.9) ǂ Biobran/MGN-3 RBCs
UA HB
AST6.0(×10(g/dL)
(1.7)
(mg/dL)(U/L) /μL)ǂ RBCsUA HB (×10 (g/dL)
12.1
4.8
0.62
(mg/dL)
7.4
17.7
6(0.5)(1.7)
/μL)
(2.9)(4.6)
WBC MCHC ALT
Neutrophils (×10 3/μL)
(g/dL) % WBC ALT
Neutrophils 57.3
(×10 22.0
(U/L)
6.8
32.3 3(1.8)
/μL)(9.3)
(14.3) %
(0.9) 52
NKT (500 mg/day)
Biobran/MGN-3 4.5 (1.6) ǂ Biobran/MGN-3
(500 RBCs HCT
mg/day)
HB 5.6 (g/dL)
(×10 (2.4)6%/μL)ǂ RBCs HB HCT 37.4
(g/dL)
12.1
(×104.8
0.44 6% (4.4)
(1.7)
(0.5)
/μL) 1
UA
LymphocytesAST
Neutrophils (mg/dL)
(U/L) % % AST
Lymphocytes
Neutrophils 32.5
57.3 7.4
17.7
(U/L) (2.9)
(4.6)
(7.9)
(14.3) % % 531
Placebo for 3 months for WBC
3 MCHC months
HCT (×10 %
3/μL)
(g/dL) MCHC HCT 37.46.8
32.3(g/dL)
% (1.8)
(0.9)
(4.4)
NK CD-107 a (500
Biobran/MGN-3 45.3 (12.0) ǂ
mg/day) (500 RBCs
Platelets
mg/day)
HB
UA 50.8(g/dL)
(×10
(19.5)
(mg/dL)
(×10 63/μL)
¥
/μL) HB187.3
UA
Platelets (g/dL)
12.14.8
0.38
(mg/dL)
7.4(7.9)
(×10 (1.7)
(0.5)
(2.9)
(37.1)
3/μL) 18 13
ALP (U/L) Lymphocytes
185.5
Neutrophils
WBC (51.3)
(×10 3/μL) %
% Lymphocytes
WBC183.2 32.5(50.1)
57.3
(×10
6.8 (14.3)
3(1.8)
/μL) % 3
for
(5003 months
Biobran/MGN-3 forMCHC
67.9 (15.6) ǂ Biobran/MGN-3
mg/day) 3 monthsHCT
HB (g/dL)
%63/μL)
(g/dL) ǂ % Platelets
MCHC HCT 32.3
37.4 (g/dL)
12.1%6(0.9)
(4.4)
3(1.7)
3
NKT CD-107 a ALT (U/L) RBCs
Platelets
22.0ALP 75.5
Lymphocytes (×10
(9.3)(U/L)
(22.3)
(×10 /μL) RBCs ALP
20.3 (×10
213.0
187.3 4.8
(U/L)
0.23
(×10
(8.4)
32.5 /μL)
(0.5)
(43.0)
(37.1)
/μL)
(7.9) 21
18
Neutrophils
WBC
MCHC (×10 3/μL)
(g/dL) % WBCNeutrophils
MCHC 57.3
(×106.8
32.3 3(1.8)
(g/dL) (14.3)
/μL)
(0.9) % 5
NK
for 3mg/day)
(500
Biobran/MGN-3
AST (U/L)
months
6.1 (2.6) ǂ Biobran/MGN-3(500 17.7ALT
ALP
Platelets HB HCT
mg/day)
6.7
(4.6)(g/dL)
(U/L)
(1.7)
(×10
% ǂ3
/μL) HB
ALT
ALP
18.3213.0 37.4
(g/dL)
12.1
22.0
(U/L)
0.60
187.3(5.8)
(4.4)
(1.7)
(7.2)
(43.0)
(37.1) 21 231
Lymphocytes
Neutrophils
WBC (×10 3 /μL) % % Lymphocytes
Neutrophils
WBC 57.3
(×10 32.5
6.8 3 (1.8)
/μL)(7.9)
(14.3) % % 5
for 3mg/day)
months for MCHC
3mg/day)
ASTmonths
HCT (g/dL)
(U/L) % ASTHCT 23.8 32.3
37.4
(U/L)% (0.9)
(4.4)
(11.0) 223
Biobran/MGN-3 NKT UA(500(mg/dL)
Biobran/MGN-3 3.1 (0.6) ǂ Biobran/MGN-3
(500 ALT
7.4
Platelets ALP (2.9)
4.6 (U/L)
(2.5)
(×10 ǂ3/μL) ALT
Platelets 8.2 22.0
(3.8)
213.0
0.25
187.3 (×10 (7.2)
(43.0)
(37.1)
3/μL)
(500 mg/day) Lymphocytes
forNeutrophils
WBCMCHC (×10 3
(g/dL) %
/μL) % Lymphocytes
Neutrophils
MCHC
32.5
57.3 6.8
32.3
(g/dL)
(7.9)
(14.3)
(1.8) %
(0.9)
% 513
NK CD-107 a RBCs for(×10
(500 3mg/day)
months
Biobran/MGN-3 6/μL)
49.5 (10.4) ǂ (500 3UA
4.8 months
AST
mg/day)
ALT
ALP
(mg/dL)
(0.5)
75.2 (U/L)
(U/L)
(6.6) ¥
UAAST4.7
ALP
(mg/dL)
23.85.9
0.004(U/L)
(0.6)
22.0
213.0 (U/L)
(2.5)
(11.0)
* (7.2)
(43.0) 223
Platelets
Lymphocytes (×103 /μL)3
% Platelets
Lymphocytes 187.3 (×103(7.9)
32.5 (37.1)
3 /μL) % post-treatmen 18
for 3 months §HBp-values
for 3 months
(g/dL) represent comparison
§for Neutrophils
WBC
p-values
3UA
12.1 months (×10
(1.7)represent
between
(mg/dL) /μL) %post-treatment
WBC
comparison UA 13.9
57.3
(×106.8
between
(mg/dL)
5.9 and
(1.6)
(14.3)
/μL)
(1.8)
(2.5) baseline values3
NKT CD-107 a (500
Biobran/MGN-3mg/day)
70.6 (10.1) ǂ Biobran/MGN-3 ALP AST
ALT
76.9 (U/L)
(U/L)
(9.8) ǂ ALT
ALP 213.023.8
22.0
(U/L)
(U/L)
0.25 (11.0)
(7.2)
(43.0) 21
because allrepresent
data werecomparisonPlatelets
normally
becauseLymphocytes
Neutrophils all (×10
data /μL)
distributed. 3
were% All Platelets
normally
comparisons
%post-treatment 187.3
Neutrophils (×10(37.1)
distributed.
32.5
57.3
3 /μL)
yielded
(7.9)
(14.3) Allnon-sign
% post-treatmencompari 18
§ HCT
p-values
for 3 %
months § p-values
37.4 UA (4.4)represent
between
(mg/dL) comparison 36.9 between
and
(3.1)
5.9 (2.5) baseline values5
§ p-values represent comparison between post-treatment and baseline (500
ment mg/day)
andallthe
values andbasal levels.(500
ment
were determined ALT
and
ALP AST
mg/day)
ǂ Comparison
using the (U/L)
(U/L)
basal
paired 3between
levels.
t-test because AST
ǂplacebo
ALT
Comparison
ALP 213.0
all 23.8
22.0
data (U/L)
(U/L)
and (11.0)
(7.2)
Biobran/MGN-3
(43.0)
were between placebo 21 2g2
because data were Platelets
normally
becauseLymphocytes all (×10
distributed.
data /μL)
were All
normally
comparisons
% post-treatment
Lymphocytes 187.3
distributed.
32.5 (37.1)
yielded
(7.9) % Allnon-sign
compari 3
MCHC
normally distributed. p ≤ 0.05 was considered statistically significant.§ * (g/dL)
p-values
The
for regards
with 3 months represent
proportion
to of 32.3
comparison
forǂ Comparison
all variables.
with 3regards (0.9)
PMA/ionomycin-stimulated
UApmonths
AST ≤ the0.05between
(mg/dL)
to
(U/L) wasallbetween
variables.NK
considered 37.4
cells
UA (1.5)
expressing
(mg/dL)
5.9
p statistically
AST ≤23.80.05
(U/L) and
was(2.5)
(11.0) baseline
considered
significant. valuesta
RB
22g
CD-107 a was significantly higher at post-treatment comparedWBC ment and
to hematocrit;
the basal
because the
levels.
allMCV,basal levels.
¥ Values
data ment
werewere
ALT
and
Platelets ALP
significantly
normally basal
(U/L)
(×10 3
higher
distributed. levels.
/μL)inAll ǂ ALT
placebo
Comparison
Platelets
the 22.0
213.0
187.3
Biobran/MGN-3
comparisons and
(×10 (7.2)
Biobran/MGN-3
between
(43.0)
(37.1)
3 /μL)
yielded placebo
non-sign 18
(×10
§ p-values 3/μL)
allmean
represent comparison 6.8
hematocrit;
§corpuscular
p-values (1.8) MCV,
tovolume;
allmean
represent
between MCH,
corpuscular
post-treatment
comparison 6.7
pmean
(1.4)
corpuscular
volume;
between
and baselineMCH, hemoglo
post-treatme mea
value
group compared to the placebo group (p = 0.026). ǂ Values werewith
Biobran/MGN-3 not
ment regards
statistically
and thetobasal variables.
different with
between
levels. UA
ǂ regards
pALT
AST (mg/dL)
≤the
Comparison
ALP
0.05
(U/L)
(U/L)was
placebo variables.
considered
and
between UAAST ≤(mg/dL)
the Biobran/MGN-3
placebo
ALP
5.9
statistically
0.05
23.8 (U/L)
22.0
213.0 and
(U/L)
(2.5)
was
(11.0)
(7.2) considered
significant.
Biobran/MGN-3
(43.0)
sta
RB
22
aminotransferase;
Neutrophils
because
hematocrit; %
allrepresent
data were
MCV, AST,
mean aminotransferase;
aspartate
57.3 (14.3)
normally
because
hematocrit;
corpuscular allaminotransferase;
distributed.
data
MCV,volume; AST,
were
mean aspartate
All
normally
MCH, UA,
60.7
comparisons
corpuscular
mean aminotransferase;
uric
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(mg/dL)
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a; NKT
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CD-107 a, PMA/ionomycin-stimulated NKT cells expressingLymphocytes ment a. and the basal levels.
ment AST
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Nutrients 2021, 13, x FOR PEER REVIEW for 3 months §hematocrit;
p-values
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to alllevels.
variables.
withcorpuscular
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Supplementation
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14
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Figure
Supplementation
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shows
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Pulmonary Epithelial BEAS-2B Cells
aminotransferase;
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§ p-values
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Figure volume;
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Biobran/MGN-3
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shows
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corpuscular
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pmean
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Biobran/MGN-3
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Figure
Supplementation corpuscular
statistically
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volume;
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and
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showsMCH,
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Significan mea
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Figure 3 shows
Figure that Biobran/MGN-3
3 shows ment
that aminotransferase;
Biobran/MGN-3 exposure
andall the basal levels.
exposure
AST, ǂ Comparison
at aminotransferase;
abecause
concentration
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concentration
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between
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of
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aminotransferase;
uric acid. Biobran/MGN-3 UA,
because
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distributed.
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Biobran/MGN-3 incidence
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ment regards
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after
concentration
aminotransferase; the all
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levels.
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intake
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ment
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Comparison
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Figure
≤Biobran/MGN-3
0.05
500
the was
intake
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Fisher’s
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considered
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20.2
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incident
Supplementation
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statistically
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te
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R
regulated the RIG-1 hematocrit; MCV, mean than
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upregulated theand MDA5
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and expression
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with regards in
to BEAS-2B
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regards
estimated ≤ Figure
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all
to 2variables.
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Supplementation
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Figure
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pared § to its expression
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2value
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exposure
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−Figure
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enhanced the expression ofaminotransferase;
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group
shows incidence
forBiobran/MGN-3
Biobran/MGN-3
RBC, compared
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Figure 2 shows the incident Biobran/MGN-3
CI 43.4
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perCIFis
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in
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using the
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Byar 81%
− estimated
95.8%).
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The the Biobran/MGN-3
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(pcompared
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the
using Biobran/MGN-3
compared
fraction
a two-tailed in to
theFi
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mated = 0.06 to be − 2.06) CICI= =0.06 tobe −2.95 2.06) − casescomparedperCI10
group was almost 82% (95% using CI =inthe
tion 9.9%
the Byar−81%
96.4%).(95%
method.
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mated
using The
Similarly, 10.5%
to
the preventive
group Byar 81%
significant 95.8%).
(95%
method.
compared fractio
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=
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(p = 0.038Fi u
when comparing the incidence mated
using to beByar
density 81%
rates (95%
inmated
theCItwo =The 10.5%
togroups.
be 81%
− 95.8%).
(95%incid
The CI =
tion inthe the Biobran/MGN-3 method. tion in the
group preventive
Biobran/MGN-
compared fract to
in the Biobran/MGN-3 group mated wastoestimated be 81%method.
(95%to be CI0.57 =the10.5% cases− per 1000 p
using the Byar using The Byar 95.8%).
preventive method. fractT
CI = 0.06 − 2.06) compared to 2.95 cases per 1000 person-days (95% CI = 1
mated to be 81% (95% matedCI = to 10.5% be 81% − 95.8%).
(95% CI
placebo group (p = 0.038 using a two-tailed Fisher’s exact test). The risk
tion in the Biobran/MGN-3 group compared to the placebo group was est
using the Byar method. The preventive fraction in the Biobran/MGN-3
mated to be 81% (95% CI = 10.5% − 95.8%).

Figure 3. Upregulation of intracellular antiviral sensors RIG-1, MDA5, and their downstream respon-
sive
Figure 3. genes ISG15 and
Upregulation ofMX1 in the human
intracellular lungsensors
antiviral BEAS-2B epithelial
RIG-1, MDA5, cellsand
when exposed
their to Biobran/MGN-3
downstream re-
sponsive genes ISG15 and MX1 in the human lung BEAS-2B epithelial cells when exposed to Bi-
obran/MGN-3 (100 μg/mL) for 72 h. Histograms depict gene expression as determined by the log
fluorescent intensity on the x-axis, whereas % of cells normalized to the maximal number on the y-
axis. At each fluorescent intensity, higher % of cells exist due to shifting of the curve to the right.
Each histogram is representative of three flow cytometry experiments.
Nutrients 2021, 13, 4133 10 of 14

(100 µg/mL) for 72 h. Histograms depict gene expression as determined by the log fluorescent
intensity on the x-axis, whereas % of cells normalized to the maximal number on the y-axis. At higher
than average fluorescent intensity, higher % of cells exist due to shifting of the curve to the right.
Each histogram is representative of three flow cytometry experiments.

4. Discussion
The current study demonstrates the safety and prophylactic efficacy of Biobran/MGN-
3 in reducing ILI incidence rate and incidence density. Supplementation of Biobran/MGN-3
(500 mg/day) for 3 months in our study did not result in any subjectively reported or
laboratory-indicated side effects. This result was consistent with safety reports obtained
from several clinical studies, with some of them reporting the safe use of Biobran/MGN-
3 at doses of 1000, 2000, and 3000 mg/kg/day [46,49,55,56]. In addition to being safe,
supplementation with Biobran/MGN-3 significantly reduced the incidence rate and risk
of ILI infection from 25.5% and 55% in the placebo group to 5% and 18%, respectively. In
support of our results, it was recently reported that ingestion of arabinoxylan (10 g/day for
5 weeks), the main constituent of Biobran/MGN-3, has reduced the incidence of common
cold and boosted the vaccine-mediated seroprotection against influenza A H1N1 compared
to the control group in seniors aged 50–79 years [57]. In our study, the preventable fraction
attributed to Biobran/MGN-3 supplementation was approximately 80%, as calculated from
a reduction of either incidence rate (considering persons only) or incidence density rate
(considering person-time).
Our finding that Biobran/MGN-3 supplementation significantly reduced ILI incidence
suggests that it could be protective against influenza as well as other viruses known to be
associated with ILI. The 80%-prevented fraction observed in the Biobran/MGN-3 group,
given that influenza viruses were isolated from only 13% of ILI cases in Egypt [8], supports
the conclusion that Biobran/MGN-3 has broad antiviral activity. Earlier studies showed
that Biobran/MGN-3 supplementation was associated with reduced risk of infection and
duration of the common cold among geriatric subjects aged 70–95 years [45]. Interestingly,
the antiviral activity of Biobran/MGN-3 has been demonstrated for non-respiratory viruses
as well. For example, Biobran/MGN-3 has been shown to inhibit replication of the HIV
virus [44] and to reduce the viral load in chronic hepatitis C patients [46].
The prophylactic activity against a broad spectrum of viruses suggests that Biobran/
MGN-3 could directly enhance the innate immune system. Therefore, we investigated
the effect of Biobran/MGN-3 on two components of the innate immune system, namely
NK cells and the intracellular viral nucleic acid receptors RIG-1 and MDA5. Our results
demonstrated that Biobran/MGN-3 supplementation significantly upregulated NK cell
activity. These results were consistent with other studies involving tissue cultures, experi-
mental animals, and humans [35–40]. NK cells play an essential role in the innate immune
response through their capability to eliminate virally-infected cells, without the need for
prior sensitization to the viral antigen [20,21,58]. Impaired NK function has been shown
to be associated with increased risk of viral infections including the influenza virus [59],
hepatitis C virus [60], and herpes viruses [61].
The effect of Biobran/MGN-3 on the expression levels of RIG-1 and MDA5, the key
intracellular sensor of viral nucleic acids, was also studied using lung epithelial cells.
Biobran/MGN-3 ingestion upregulated the expression of both receptors, which was func-
tionally relevant, as confirmed by the upregulated expression of their downstream targets
ISG15 and MX1. RIG-I was found to be activated by positive- and negative-stranded RNA
viruses such as influenza, Rift Valley fever, measles, Ebola, vesicular stomatitis, and hepati-
tis C viruses [62], whereas MDA5 was found to be activated by picornavirus, arteriviruses,
hepatitis D, encephalomyocarditis, and Kaposi’s sarcoma-associated herpesvirus [63–66].
Our finding that ISG15 has been upregulated is important because it has been shown to
inhibit replication of several viruses such as the influenza virus, vaccinia virus, human im-
munodeficiency virus 1, vesicular stomatitis virus, and dengue virus [18,67]. Homozygous
ISG15-knockout mice exhibited high mortality rate when infected with influenza B or A
Nutrients 2021, 13, 4133 11 of 14

viruses [18,19]. Similarly, MX1 upregulation is also important because it has been linked to
the high resistance of the A2G mouse inbred strain to influenza infection [17]. Collectively,
Biobran/MGN-3 was found to upregulate the expression of RIG-1 and MDA5 intracellular
receptors and their downstream ISG15 and MX1, with known direct and indirect broad
antiviral activity.
The finding of the current study has several implications. One implication is related
to NK induction. Several lines of research have focused on NK cells as a prophylactic
and/or therapeutic agent against virus infection and cancers [20–24]. In this regard,
Bioran/MGN-3 could be a novel agent for modulating NK activity. Another implication
is related to the induction of RIG-1 and MDA-5. A crucial role for MDA5 and RIG-1 in
sensing and mounting effective antiviral response against SARS-Cov-2 has recently been
reported [68,69]. Therefore, it is plausible to propose that Biobran/MGN-3 could counteract
COVID-19 infection. It is worth mentioning that Biobran/MGN-3 is a notable biological
response modifier in that it does not exhibit hyporesponsiveness [48], making it a unique
and attractive agent for long-term preventive and/or therapeutic purposes.
The strengths of our study stem from (1) its elimination of biased assessment because
of its design as a prospective double-blind placebo-controlled clinical trial, (2) its emphasis
on the prophylactic efficacy and safety of Biobran/MGN-3, and (3) its inclination towards
older individuals (average age of 60 years) who are more susceptible to infection and
cancers. The study is nevertheless limited in its relatively small sample size, assessment
of RIG-1, MDA5, ISG-15, and MX1, which was performed in tissue culture, and lack of
disease severity assessment after infection. The results are very encouraging and warrant
replications with larger sample sizes and more detailed assessments.

5. Conclusions and Future Direction

Our data suggest that Biobran/MGN-3 is a safe, non-toxic, and effective antiviral
agent whose activity stems from its potent stimulating effect on several components of the
innate immune system. Biobran/MGN-3 supplementation was found to upregulate NK
cell activity and the expression levels of RIG-1 and MDA5, as well as their downstream
ISG15 and MX1 genes, known for their broad antiviral effects. Biobran/MGN-3 could be a
novel antiviral agent that is well suited for long-term preventive purposes. Our future work
includes inclusion of a larger sample size and expands our objective to include antiviral
effects against COVID-19.

Author Contributions: Conceptualization, A.F.E., S.A., A.A. and M.G.; Data curation, A.F.E., S.A.,
A.A. and M.G.; Formal analysis, A.F.E., S.A., A.A. and M.G.; Funding acquisition, M.G.; Investigation,
A.F.E., S.A., A.A. and M.G.; Methodology, A.F.E. and M.G.; Resources, M.G.; Writing—original draft,
A.F.E. and M.G.; Writing—review and editing, A.F.E. and M.G. All authors will be informed about
each step of manuscript processing including submission, revision, revision reminder, etc., via emails
from our system or assigned Assistant Editor. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study protocol conformed to the ethical guidelines of
the 1975 Declaration of Helsinki and was approved by Institutional Review Board, Zagazig University
Hospital, Faculty of Medicine (IRB# 1507/1/6/2017).
Informed Consent Statement: Informed consents were obtained from willing participants after
explaining the study purpose and design.
Data Availability Statement: Data are available upon request from the corresponding author or
Ghoneum M.
Acknowledgments: The authors gratefully acknowledge Daiwa Pharmaceutical Co., Ltd. for provid-
ing Biobran/MGN-3.
Conflicts of Interest: The authors declare no conflict of interest.
Nutrients 2021, 13, 4133 12 of 14

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