In i o and in i o e ec s o
Pela gonium sidoides DC. oo
ex ac EPs
®
7630 and selec ed
cons i uen s agains
SARS-CoV-2 B.1, Del a AY.4/
AY.117 and Omic on BA.2
Jackson Emanuel
1
,
2†
, Jan Papies
1
,
2†
, Celine Galande
1
,
2†
,
Julia M. Adle
3
, Nicolas Heinemann
1
,
2
, Ka h in Eschke
3
,
Sophie Me z
4
, Hannah Pischon
4
, Ruben Rose
5
, Andi K umbholz
5
,
6
,
Ža ko Kulić
7
, Ma in D. Lehne
7
, Jakob T impe
3‡
and
Ma cel A. Mülle
1
,
2
*
‡
1
Ins i u e o Vi ology, Cha i é—Uni e si ä smedizin Be lin, Co po a e Membe o F eie Uni e si ä Be lin,
Humbold -Uni e si ä zu Be lin, Be lin, Ge many,
2
Ge man Cen e o In ec ion Resea ch (DZIF), Pa ne
Si e Cha i é, Be lin, Ge many,
3
Ins i u ü Vi ologie, F eie Uni e si ä Be lin, Be lin, Ge many,
4
IDEXX
Labo a o ies, Ko nwes heim, Ge many,
5
Ins i u e o In ec ion Medicine, Kiel Uni e si y and Uni e si y
Hospi al Schleswig-Hols ein, Kiel, Ge many,
6
Labo D . K ause und Kollegen MVZ GmbH, Kiel, Ge many,
7
P eclinical R&D, D . Willma Schwabe GmbH and Co. KG, Ka ls uhe, Ge many
The occu ence o immune-e asi e SARS-CoV-2 s ains emphasizes he impo ance o
sea ch o b oad-ac ing an i i al compounds. Ou p e ious in i o s udy showed ha
Pela gonium sidoides DC. oo ex ac EPs
®
7630 has combined an i i al and
immunomodula o y p ope ies in SARS-CoV-2-in ec ed human lung cells. He e we
assessed in i o e ec s o EPs
®
7630 in SARS-CoV-2-in ec ed hams e s, and
in es iga ed p ope ies o EPs
®
7630 and i s unc ionally ele an cons i uen s in
con ex o pheno ypically dis inc SARS-CoV-2 a ian s. We show ha EPs
®
7630 educed i al load ea ly in he cou se o in ec ion and displayed significan
immunomodula o y p ope ies posi i ely modula ing disease p og ession in
hams e s. In addi ion, we find ha EPs
®
7630 di e en ially inhibi s SARS-CoV-
2 a ian s in nasal and b onchial human ai way epi helial cells. An i i al e ec s we e
mo e p onounced agains Omic on BA.2 compa ed o B.1 and Del a, he la e wo
p e e ing TMPRSS2-media ed usion wi h he plasma memb ane o cell en y ins ead
o ecep o -media ed low pH-dependen endocy osis. By using SARS-CoV-2 Spike
VSV-based pseudo pa icles (VSVpp), we confi m highe EPs
®
7630 ac i i y agains
Omic on Spike-VSVpp, which seems independen o he se ine p o ease TMPRSS2,
sugges ing ha EPs
®
7630 a ge s endosomal en y. We iden i y a leas wo molecula
cons i uen s o EPs
®
7630, i.e., (−)-epigalloca echin and axi olin wi h an i i al e ec s on
SARS-CoV-2 eplica ion and cell en y. In summa y, ou s udy shows ha EPs
®
7630 amelio a es disease ou come in SARS-CoV-2-in ec ed hams e s and has
enhanced ac i i y agains Omic on, appa en ly by limi ing la e endosomal SARS-
CoV-2 en y.
OPEN ACCESS
EDITED BY
Thomas B endle ,
Plan aPhile
®
, Uni ed S a es
REVIEWED BY
Klaus Pe e La é,
Independen esea che , Ge many
Benjamin Ki chwege ,
Uni e si y o Vienna, Aus ia
*CORRESPONDENCE
Ma cel A. Mülle ,
[email p o ec ed]
†
These au ho s ha e con ibu ed equally
o his wo k and sha e fi s au ho ship
‡
These au ho s ha e con ibu ed equally
o his wo k
RECEIVED 29 Ap il 2023
ACCEPTED 11 July 2023
PUBLISHED 26 July 2023
CITATION
Emanuel J, Papies J, Galande C,
Adle JM, Heinemann N, Eschke K,
Me z S, Pischon H, Rose R, K umbholz A,
KulićŽ, Lehne MD, T impe J and
Mülle MA (2023), In i o and in i o
e ec s o Pela gonium sidoides DC. oo
ex ac EPs
®
7630 and selec ed
cons i uen s agains SARS-CoV-2 B.1,
Del a AY.4/AY.117 and Omic on BA.2.
F on . Pha macol. 14:1214351.
doi: 10.3389/ pha .2023.1214351
COPYRIGHT
© 2023 Emanuel, Papies, Galande , Adle ,
Heinemann, Eschke, Me z, Pischon, Rose,
K umbholz, Kulić, Lehne , T impe and
Mülle . This is an open-access a icle
dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License
(CC BY). The use, dis ibu ion o
ep oduc ion in o he o ums is
pe mi ed, p o ided he o iginal au ho (s)
and he copy igh owne (s) a e c edi ed
and ha he o iginal publica ion in his
jou nal is ci ed, in acco dance wi h
accep ed academic p ac ice. No use,
dis ibu ion o ep oduc ion is pe mi ed
which does no comply wi h hese e ms.
F on ie s in Pha macology on ie sin.o g01
TYPE O iginal Resea ch
PUBLISHED 26 July 2023
DOI 10.3389/ pha .2023.1214351
KEYWORDS
SARS-CoV-2, co ona i us, Pela gonium sidoides, EPs 7630, d ug epu posing, immune
modula ion, COVID-19, cy okine s o m
1 In oduc ion
Since i s in oduc ion in o humans in la e 2019 and global
sp ead h oughou 2020, SARS-CoV-2 has become endemic in
he human popula ion and emains an impo an public heal h
challenge. Successi ely eme gen SARS-CoV-2 a ian s,
including Del a (e.g., AY.4, AY.117) and Omic on (e.g., BA.1,
BA.2, BA2.75, XBB.1.5), ha e p ope ies ha con e esis ance
o exis ing an i i al he apies. Specifically, accines and
monoclonal an ibody he apies, which p e iously elici ed
s ong neu aliza ion o SARS-CoV-2, show significan ly
educed neu aliza ion o cu en ly ci cula ing a ian s
(Da is e al., 2021;Eda a e al., 2022;Tada e al., 2022;
Wang e al., 2023). Remaining me hods o con olling
SARS-CoV-2/COVID-19 show a ying deg ees o e ficacy
(Mohamed e al., 2022), and may be a ec ed by he
eme gence o u u e SARS-CoV-2 a ian s. This emphasizes
he con inuing need o new o e-pu posed, b oad-ac ing
an i i al he apeu ics ha can o e come i al esis ance.
COVID-19 can esul in se e e inflamma o y esponses and
immunological dys egula ions. The elease o p o-inflamma o y
cy okines and infil a ion o immune cells in o he lungs a e key
cha ac e is ics o se e e COVID-19-associa ed acu e
espi a o y dis ess synd ome (Pelaia e al., 2020;Hu e al.,
2021;Hönzke e al., 2022). Se e al COVID-19- ela ed immune
ma ke s we e p oposed as po en ial d uggable a ge s in he
ea men o COVID-19, including membe s o he CXC
chemokine amily ha p omo e immune cell chemo axis o
he si e o in ec ion (Didangelos, 2020;Gudowska-Sawczuk
and M oczko, 2022). The apeu ics which educe he hos
p o-inflamma o y esponse by limi ing he elease o
cy okines, such as IL-8, CXCL9, and IP-10 (Zhao e al., 2020;
Callahan e al., 2021;Ma sushima e al., 2022) may he e o e
educe excessi e immune cell infil a ion o he lungs and
imp o e hos espi a o y unc ion. An i-SARS-CoV-
2 he apeu ics should ideally combine an i i al and
immunomodula o y p ope ies, so ha bo h he i us and
he symp oms/disease can be e ec i ely a ge ed wi h a single
ea men .
Pela gonium sidoides DC. is a medicinal plan indigenous o
Sou h A ica (B endle and an Wyk, 2008). In a p e ious s udy,
we cha ac e ized he P. sidoides DC. oo ex ac EPs
®
7630, and
ound ha i has bo h an i i al and an i-inflamma o y e ec s on
SARS-CoV-2 in ec ion in i o (Papies e al., 2021), making i a
sui able candida e o u he p eclinical in i o model-based
in es iga ions. The Sy ian hams e is a widely accep ed model
ha allows o ully e alua e COVID-19 and i s e ec s on disease
pa hology connec ed o a dys egula ed immune esponse
(Os e iede e al., 2020). Howe e , de ailed in es iga ions o
immunomodula o y e ec s in hams e models a e challenging
due o a lack o alida ed immunological ools. Sophis ica ed
human espi a o y in i o models migh se e as a
complemen a y model o in es iga e pu a i e an i i al and
an i-inflamma o y e ec s o immunomodula o y compounds
du ing SARS-CoV-2 in ec ions a he ac ual si e o eplica ion.
EPs
®
7630, a p op ie a y hyd oe hanolic ex ac o P.
sidoides DC. (Ge aniaceae) oo s, is he ac i e p inciple in
he bal medicinal p oduc s used o he ea men o
espi a o y ac in ec ions such as acu e b onchi is o
common cold (Ma hys e al., 2003;Chuchalin e al., 2005;
Kamin e al., 2010a;Kamin e al., 2010b;Riley e al., 2019). I is
composed o ca bohyd a es, mine als, pep ides, pu ine
de i a i es, highly subs i u ed benzopy anones, and oligo-
and polyme ic p odelphinidins (Schö z e al., 2008).
Sub ac iona ion o mul icomponen en i ies o
pha macological es ing can help o dis inguish an i i al and
immunomodula o y e ec s o plan ex ac s and can be ca ied
ou ollowing di e en s a egies. EPs
®
7630 sub ac ions es ed
by Papies e al. we e gene a ed by ul afil a ion, gene a ing
ac ions con aining molecules o di e en molecula sizes. In
con as , an o hogonal s a egy o sub ac iona ion in ol es
selec ing single cha ac e is ic molecules o EPs
®
7630 ep esen ing di e en na u al p oduc classes. Fo
example, umckalin and umckalin sul a e a e cha ac e is ic
TABLE 1 EPs
®
7630 small molecules.
Compound (cpd) Name Con en in EPs
®
7630 (m/m) Subs ance class
cpd A umckalin ~0.2–0.8% in ba ch 878
a
benzopy anone
cpd B umckalin sul a e ~0.2–0.8% Σ= 0.75% benzopy anone
cpd C (−)-epigalloca echin <0.3% ca echin
cpd D p odelphinidin B1 <0.7% p odelphinidin
cpd E p odelphinidin B4 <0.7% p odelphinidin
cpd F (−)-epigalloca echin galla e no analyzed
b
ca echin
cpd G (+)- axi olin ~0.1% (Kuliće al., 2022)fla onoid
a
As specified in he ce ifica e o analysis o he ba ch. The con en s o he ca echins and p odelphinidins we e calcula ed om isola ion yields (da a no shown).
b
(−)-epigalloca echin galla e has been desc ibed o a 50% me hanolic Pela gonium sidoides ex ac (Sa ickiene e al., 2018). Quan ifica ion o gallic acid a e hyd olysis o ca. 0.01% in EPs®
7630 may co espond o a possible con en o ca. <0.027% epigalloca echin galla e.
F on ie s in Pha macology on ie sin.o g02
Emanuel e al. 10.3389/ pha .2023.1214351
ep esen a i es o benzopy anones (couma ins) ound in EPs
®
7630. Taxi olin sul a e and o he fla onoid sul a es we e
ecen ly disco e ed as genuine cons i uen s in P. sidoides
oo ex ac EPs
®
7630 (Kuliće al., 2022). Taxi olin has been
iden ified in silico as a po en ial inhibi o o SARS-CoV-
2p o ease(Fische e al., 2020) and RNA-dependen RNA
polyme ase (Kandeel e al., 2020). In addi ion, ca echins such
as galloca echin, epigalloca echin and epigalloca echin galla e
we e iden ified in EPs
®
7630. Epigalloca echins om g een ea,
o example, we e p e iously shown o inhibi SARS-CoV-2 and
o he CoV en y (Henss e al., 2021;Liu e al., 2021;Ohishi e al.,
2022). Fu he mo e, EPs
®
7630 con ains p odelphinidin B1
(Epigalloca echin-4β→8-galloca echin) and p odelphinidin
B4 (Galloca echin-4α→8-epigalloca echin) which a e dime ic
p odelphinidins co e ing di e en s e eochemical
configu a ions o his subs ance class. Al hough mul iple
sub ac ions o EPs
®
7630 we e ound o con ibu e o i s
ac i i y (Papies e al., 2021), he ela i e con ibu ion o
indi idual molecula componen s o EPs
®
7630 has no ye
been de e mined. Since SARS-CoV-2 a ian Omic on has
been shown o p e e an al e ed cellula en y mechanism
compa ed o p eceding a ian s (Meng e al., 2022;Wille
e al., 2022), i may be he case ha en y- a ge ing
compounds exhibi di e en ial ac i i y agains SARS-CoV-
2 a ian s. Ini ial epo s sugges ha Omic on exhibi s less
TMPRSS2-dependen plasma memb ane usion and a o s
TMPRSS2-independen la e endosomal en y (Meng e al.,
2022;Wille e al., 2022). Thus, examining he deg ee o
which EPs
®
7630 and i s componen s inhibi he en y o
pheno ypically di e en SARS-CoV-2 a ian s p o ides a
me hod o s udying he an i i al mechanism o EPs
®
7630 in
g ea e de ail.
FIGURE 1
EPs
®
7630 has limi ed e ec s on i us eplica ion in i o.(A) Schema ic ea men o e iew. Hams e s ecei ed Pela gonium sidoides oo ex ac
ei he o ally (p.o.) o bo h o ally and in anasally (p.o. + i. n.). On day 0 animals we e in ec ed wi h 10
5
PFU SARS-CoV-2 B.1 a ian eu hanized on day 2,
4 and 7 a e in ec ion. (B) Genomic i al RNA copies and (C) PFU pe swab. (D) Genomic i al RNA copies and (E) PFU in homogenized lung issue.
S a is ical significance is indica ed by (*) as de e mined by wo-way ANOVA o he log- ans o med da a wi h Dunne ’s mul iple compa ison es .
As e isks a e shown only o significan ly di e en da a se s. (*) = p<0.05; (**) = p<0.01; (***) = p<0.001.
F on ie s in Pha macology on ie sin.o g03
Emanuel e al. 10.3389/ pha .2023.1214351
2 Ma e ials and me hods
2.1 Pela gonium sidoides DC. Ex ac EPs
®
7630 and indi idual cons i uen s
Fo all expe imen s, a sample o a p oduc ion ba ch (EXCh.
878) o EPs
®
7630, a d ied ex ac o P. sidoides DC. oo s (1:
8–10), ex ac ion sol en : e hanol 11% (w/w) was used. 80% o
he oo s used o he a o emen ioned p oduc ion ba ch we e
collec ed om wild plan popula ions and 20% we e ha es ed
om plan a ions in Sou h A ica. P io o ex ac ion, he d ied
plan ma e ial was es ed in an a ay o DNA-based and
phy ochemical me hods o confi m he quali y and iden i y
o he he bal ma e ial. Pha macognosy was done by he
quali y con ol depa men o D . Willma Schwabe GmbH
and Co. KG. Vouche specimens o e e y lo a e deposi ed in
he Depa men o Pha macognosy o be e ained o 10 yea s.
Chemical finge p in ing o he used EPs
®
7630 ba ch acco ding
o he Consensus s a emen on he Phy ochemical
Cha ac e iza ion o Medicinal Plan ex ac s (Hein ich e al.,
FIGURE 2
His opa hology e eals significan e ec s o EPs
®
7630 on lungs o SARS-CoV-2-in ec ed hams e s. His opa hology o ep esen a i e hema oxylin-
and-eosin-s ained, pa a fin-embedded le lungs compa ing he h ee g oups ( ehicle, EPs
®
7630 p. o., EPs
®
7630 i. n. and p. o.) in pa ame e s b onchi is
and edema. (A) Se e e b onchi is in he con ol g oup ( ehicle, le column), indica ed by la ge amoun s o neu ophils in he b onchial lumen (a ow) as
well as b onchial epi helial cell nec osis. Delayed onse o b onchi is in he EPs
®
7630 i. n. and p. o. g oup ( igh column) wi h occu ence o
neu ophil infil a ion in b onchial lumina (a ow) by day 4 o in ec ion. All g oups show p oli e a i e egene a i e change o he b onchial epi helium wi h
b onchial epi helial hype plasia in he la e s age (as e isk) by day 7 o in ec ion (bo om ow). (B) Pe i ascula and al eola edema o ma ion less p ominen
in he EPs
®
7630 i. n. and p. o. g oup in compa ison o ehicle and EPs
®
7630 p. o. g oup (middle ow, day 4 o in ec ion). P ominen egene a i e change in
all g oups du ing he la e s age (day 7 o in ec ion) wi h s ong pneumocy e ype 2 hype plasia (hash symbol) (bo om ow). Scale ba 100 µm o all
pic u es.
F on ie s in Pha macology on ie sin.o g04
Emanuel e al. 10.3389/ pha .2023.1214351
2022) by h ee di e en me hods (NMR, HPLC, GPC) was
al eady ca ied ou in ou p e ious s udy (Papies e al., 2021).
Umckalin (7-Hyd oxy-5,6-dime hoxy-2H-1-benzopy an-2-
one, cpd A) (Table 1) and umckalin sul a e (5,6-Dime hoxy-7-
(sul ooxy)-2H-1-benzopy an-2-one, cpd B) we e isola ed as
desc ibed p e iously (Schö z e al., 2008). (−)-Epigalloca echin
(cpd C) was pu chased om In e chim S.A., F ance.
P odelphinidin B1 (Epigalloca echin-4β→8-galloca echin, cpd
D) and p odelphinidin B4 (Galloca echin-4α→8-
epigalloca echin, cpd E) we e isola ed by ac iona ion o he
low molecula weigh p odelphinidin ac ion con aining dime s
and ime s, as p e iously desc ibed (Schö z and Nöldne , 2007).
The single compounds we e pu ified by dissol ing he
a o emen ioned dime / ime ac ion in me hanol o a
concen a ion o 12% (w/ ) and loading he solu ion on a
Toyopea l HW-40S column (leng h = 45 cm, diame e =
2.5 cm) p econdi ioned wi h me hanol, which was p e iously
sa u a ed wi h N
2
gas. F ac ions we e elu ed om he esin by
an isoc a ic N
2
-sa u a ed me hanol flow. F ac ions we e checked
by hin laye ch oma og aphy and pooled o yield he single
compounds. (−)-Epigalloca echin galla e (cpd F) was pu chased
om TCI Deu schland GmbH, Ge many. (+)-Taxi olin (cpd G)
was pu chased om Me ck KGaA, Da ms ad , Ge many. The
pu i y o he pu chased compounds we e aken om he endo s’
specifica ions and had an >98% HPLC pu i y. The pu i y o he
isola ed subs ances was checked by
1
H-NMR spec oscopy
(Supplemen a y Figu e S1), using he same ins umen as
desc ibed p e iously (Papies e al., 2021). Apa om esidual
sol en s (wa e , e hanol), no addi ional impu i ies could be
de ec ed o umckalin and umckalin sul a e in he NMR
spec a. Fo p odelphinidin B1 and B4, some mino impu i ies
om oligo-/polyme ic p odelphinidins we e de ec able as signal
bulges below he sha p signals o he espec i e pu e dime , in
addi ion o esidual sol en (wa e ). Thus, he pu i y o all
subs ances can be conside ed sui able o he s udy.
Tes solu ions we e p epa ed as ollows: EPs
®
7630 was suspended in
DMEM o a s ock concen a ion o 2 mg/mL, and se ially dilu ed in
assay medium o achie e he equi ed wo king concen a ion. All
compounds lis ed in Table 1 we e p epa ed by suspension in DMSO
o s ock concen a ions o 10 mg/mL, and se ially dilu ed in assay
medium o achie e he equi ed wo king concen a ion. DMSO
ehicle con ols con ained an equi alen amoun o DMSO o he
amoun o DMSO in wells ea ed wi h he highes concen a ion o
compound in each assay.
FIGURE 3
EPs
®
7630 delays b onchioli is and limi s lung edema in SARS-CoV-2-in ec ed hams e s. Hams e s ecei ed Pela gonium sidoides oo ex ac
ei he o ally (p.o.) o bo h o ally and in anasally (p.o. + i. n.) as desc ibed in Figu e 1.(A) App oxima e lung a ea a ec ed by inflamma o y damage in
pe cen age pe g oup and ime poin . (B–D) Semi-quan i a i e sco ing o pneumonia se e i y (B) b onchi is (C) and Edema (pe i ascula and al eola ) (D)
o all g oups and espec i e ime poin s. S a is ical significance is indica ed by (*) as de e mined by wo-way ANOVA o he da a wi h Dunne ’s
mul iple compa ison es . As e isks a e shown only o significan ly di e en da a se s. (*) = p<0.05; (**) = p<0.01; (***) = p<0.001; (****) = p<0.0001.
F on ie s in Pha macology on ie sin.o g05
Emanuel e al. 10.3389/ pha .2023.1214351

2.2 E hics s a emen
In i o expe imen s we e pe o med in he biosa e y le el h ee
(BSL-3) acili y a he Ins i u ü Vi ologie, F eie Uni e si ä Be lin,
Ge many. Animal wo k was app o ed and execu ed in compliance
wi h all applicable ins i u ional, na ional and in e na ional
egula ions (Landesam ü Gesundhei und Soziales Be lin,
pe mi numbe 0086/20).
2.3 Animal husband y
Sy ian hams e s (Mesoc ice us au a us; b eed RjHan:AURA) we e
pu chased om Jan ie Labs a 10 weeks o age. The animals we e kep in
indi idually en ila ed cages (IVCs) in g oups o 1–3hams e sandhad
1 week o ge used o he housing condi ions. Food and wa e we e o e ed
ad libi um. Du ing he expe imen , he cage empe a u e was cons an ly
be ween 22°Cand24
°C wi h a ela i e humidi y be ween 40% and 55%.
2.4 In ec ion expe imen s
Sy ian hams e s we e andomly assigned in o g oups o
9 animals (40%–60% emale hams e s pe g oup). In anasal
in ec ion wi h 10
5
plaque o ming uni s (PFU) o SARS-CoV-2
(Be aCoV/Munich/Ba Pa 1/2020) in 60 µL minimal essen ial
medium (MEM) was pe o med unde gene al anes hesia. EPs
®
7630 was applied in s awbe y sy up o ally a a dose o
50 mg/kg body weigh wice daily. One ea men g oup ecei ed
he fi s dosage o EPs
®
7630 1 day be o e in ec ion, while he second
he apeu ic and ehicle ea men g oup we e s a ed on he day o
in ec ion. The ehicle g oup ecei ed s awbe y sy up wi hou EPs
®
7630. The ea men g oup ha s a ed on he day o in ec ion
addi ionally ecei ed EPs
®
7630 in anasally a 5 mg/mL oge he
wi h he i us inoculum (60 µL o al olume).
The a ionale o include an addi ional in anasal adminis a ion
o EPs
®
7630 in one o he ea men g oups was based on esul s
om ou p e ious s udy (Papies e al., 2021). In ha s udy
ac iona ion o EPs
®
7630 demons a ed highes an i i al ac i i y
in ac ions con aining oligome ic p oan hocyanidins wi h expec ed
low o al bioa ailabili y. We assumed ha local adminis a ion a he
si e o in ec ion o bypass low sys emic bioa ailabili y could inc ease
an i i al ac i i y. We decided o include a single in anasal
adminis a ion concomi an ly wi h he i us inoculum as a fi s
p oo -o -p inciple app oach o assess whe he opical mucosal
adminis a ion holds any p omise as a u u e de elopmen op ion.
In ec ed hams e s we e checked wice daily o de elopmen o
clinical symp oms and body weigh loss. Eu hanasia was scheduled
on day 2, 4 and 7 a e in ec ion. Animals we e anes he ized wi h
mede omidine (0.15 mg/kg body weigh ), midazolam (2 mg/kg body
FIGURE 4
SARS-CoV-2 (B.1) p opaga ion and inflamma o y cy okine exp ession in human b onchial ai way epi helial cells (bAEC). (A) B onchial AEC we e
inocula ed wi h SARS-CoV-2 (MOI = 0.005) wi h and wi hou EPs
®
7630 (100 μg/mL) ea men a 37°C o 2 h. Fo sample collec ion, he apical side o
he bAEC was incuba ed wi h 250 µL Mucilai medium o 20 min, which was subsequen ly emo ed and ozen a −80°C un il analysis. Supe na an s
we e analyzed by plaque assays be ween 0 and 72 h pos -in ec ion (B) o a 24 h pos -in ec ion using he Human Cy okine/Chemokine/G ow h
Fac o Mul iplex Assay (Me ck Millipo e) wi h he Luminex MAGPIX Sys em acco ding o he manu ac u e ’s ins uc ions (C). Da a a e de i ed om n=
3 biological samples. Cell cul u e medium was used as he ehicle con ol. No s a is ical significance was obse ed o (B), as de e mined by wo-way
ANOVA wi h Tukey’s mul iple compa ison es on log- ans o med da a. S a is ical significance o (C) was de e mined by pai ed - es s. As e isks a e
shown only o significan ly di e en da a se s. (*) = p<0.05. ALI = ai liquid in e ace.
F on ie s in Pha macology on ie sin.o g06
Emanuel e al. 10.3389/ pha .2023.1214351
weigh ), and bu o phanol (2.5 mg/kg body weigh ) p io o
eu hanasia. Lungs, se um, EDTA blood and o opha yngeal swabs
we e collec ed o conduc i ological and his opa hological analysis.
2.5 RNA ex ac ion and qPCR
RNA was ex ac ed om o opha yngeal swabs and 25 mg
homogenized lung issue using innuPREP Vi us DNA/RNA Ki
(Analy ic Jena, Jena, Ge many) acco ding o he manu ac u e ’s
ins uc ions. NEB Luna uni e sal P obe One-S ep RT-qPCR Ki
(New England Biolabs, Ipswich, MA, Uni ed S a es) was used o
pe o m qPCR wi h cycling condi ions o 10 min a 55°C o
e e se ansc ip ion, 3 min a 94°C o ac i a ion o he enzyme,
and 40 cycles o 15 s a 94°Cand30sa 58
°ConaqTowe
G3 cycle (Analy ic Jena, Jena, Ge many) in sealed qPCR 96-well
pla es. To moni o i us g ow h, SARS-CoV-2 RNA was
quan ified in cell cul u e supe na an s by RT-qPCR a ge ing
he SARS-CoV-2 E gene, as desc ibed p e iously (Co man e al.,
2020).
2.6 Plaque assay o in i o expe imen s
To quan i y eplica ion-compe en in ec ious i us, i a ions
we e pe o med om 50 mg lung issue and o opha yngeal
swabs. Fo sample p epa a ion, swabs we e hawed, kep in
i us anspo medium (PBS wi h 25 mg/L en ofloxacin and
10 mg/L o iconazole) o 30 min and o exed 3 imes du ing
incuba ion. The o gan samples we e homogenized in a bead mill
p ocedu e wi h ce amic beads (Analy ic Jena). The ea e , 10-
old se ial dilu ions we e p epa ed s a ing om −1 o−6and
pla ed on Ve oE6 cells g own in 12-well pla es. The pla es we e
incuba ed o 2 h a 37°C and subsequen ly o e laid wi h MEM
medium con aining 1.5% ca boxyme hylcellulose sodium (Sigma
Ald ich, S . Louis, MO, Uni ed S a es). The pla es we e fixed wi h
4% PBS-bu e ed o maldehyde solu ion 72 h a e in ec ion.
0.75% me hylene blue was used o isualize and manually
coun plaques. The assay-specific limi o de ec ion is 10 PFU/
50 mg issue. All i a ion expe imen s we e pe o med in
duplica e wells. Fo samples wi hou de ec able plaques, a
alue o 5 PFU, co esponding o hal he assay limi o
de ec ion was assigned o allow log- ans o ma ion o da a.
2.7 His opa hology
The le lung lobe was p epa ed o his opa hological
examina ion as p e iously desc ibed (Os e iede e al., 2020).
A e ca e ul p epa a ion, i was fixed in PBS-bu e ed 4%
o maldehyde solu ion o 48 h, embedded in pa a fin and cu a
2μm hickness. Subsequen ly, he slides we e s ained wi h
hema oxylin and eosin (H&E) as p e iously published (Be zbach
e al., 2021).
FIGURE 5
Enhanced inhibi ion o SARS-CoV-2 a ian BA.2 by EPs
®
7630 in human b onchial ai way epi helial cells (bAEC). Nasal AEC (A, C) and bAEC (B, D)
we e inocula ed wi h SARS-CoV-2 a ian AY.4 (A, B) o BA.2 (C, D) using an MOI o 0.005 wi h and wi h EPs
®
7630 (100 μg/mL) ea men a 37°C o 2h.
Fo sample collec ion, he apical side o he AEC was incuba ed wi h 250 µL Mucilai medium o 20 min, which was subsequen ly emo ed and ozen
a −80°C un il analysis. Supe na an s we e analyzed by plaque assays be ween 0 and 72 h pos -in ec ion. Da a a e de i ed om n= 3 biological
samples. Va ian g ow h kine ics a e shown in pa allel wi h he B.1 g ow h kine ics p e iously depic ed in Figu e 4B. Cell cul u e medium was used as he
ehicle con ol. S a is ical significance was de e mined by wo-way ANOVA wi h Tukey’s mul iple compa ison es on log- ans o med da a. As e isks a e
shown when EPs
®
7630 ea men esul ed in significan ly di e en le els o Del a o Omic on PFU o gi en imepoin s. (*) = p<0.05; (**) = p<0.01;
(***) = p<0.001; (****) = p<0.0001.
F on ie s in Pha macology on ie sin.o g07
Emanuel e al. 10.3389/ pha .2023.1214351
2.8 Cell lines
Calu-3 (ATCC HTB-55), Ve oFM (ATCC CCL-81), A549-ACE2,
A549-ACE2-TMPRSS2 (Wide a e al., 2021) we e g own in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemen ed wi h 10% e al bo ine
se um (FBS), 1% non-essen ial amino acids, and 1% sodium py u a e a
37°Cand5%CO
2
. Ve oE6 (ATCC CRL-1586) and Ve oE6-TMPRSS2
(NIBSC 100978) cells we e cul u ed in minimal essen ial medium
(MEM) con aining 10% e al bo ine se um, 100 IU/mL penicillin G,
and 100 μg/mL s ep omycin. Fo Ve oE6-TMPRSS2 cell cul u e
(NIBSC 100978), he medium also con ained 1,000 μg/mL gene icin
(G418) o selec o cells exp essing TMPRSS2. The cells we e incuba ed
a 37°Cand5%CO
2
. All cell lines we e cul i a ed unde s e ile labo a o y
condi ions and es ed o simian i us 5 and mycoplasma con amina ion
as desc ibed p e iously (Biesold e al., 2011).
2.9 Vi us s ains and in ec ion
The SARS-CoV-2 s ain Munich/2020/984 was isola ed om a
espi a o y swab ob ained om he ea ly 2020 Munich pa ien coho
(GenBank: MT270101; GISAID: EPI_ISL_406862). The Del a
FIGURE 6
Di e en ial en y inhibi ion o SARS-CoV-2 a ian s by EPs
®
7630. All cells we e p e- ea ed wi h he indica ed compounds o 2 h p e-in ec ion a
37°C. In ec ion o Calu-3, A549-ACE2, and A549-ACE2/TMPRSS2 cells wi h VSV-G as con ol (A), SARS-CoV-2-S VSVpp (SARS-CoV-2-S) om B.1 (B),
a ian Del a AY.117 (C), o a ian Omic on BA.2 (D) was done in he p esence o compounds o 30 min a 4°C a 300 g ollowed by 1-h incuba ion a
37°C. The medium was hen eplaced by DMEM con aining he indica ed compounds. DMSO was addi ionally used as a ehicle con ol. As posi i e
con ols, we applied 1 and 10 µM niclosamide (pH-dependen endosomal en y inhibi o ) and 1 and 10 µM camos a mesyla e (TMPRSS2 inhibi o ). Cell
lysa es we e p epa ed a e 24 h and he luci e ase signal was measu ed using a mul i-mode 96-well pla e eade . S a is ical significance is indica ed by (*)
as de e mined by wo-way ANOVA o he da a wi h Dunne ’s mul iple compa ison es . As e isks a e shown only o significan ly di e en da a se s. (*) =
p<0.05; (**) = p<0.01; (***) = p<0.001; (****) = p<0.0001.
F on ie s in Pha macology on ie sin.o g08
Emanuel e al. 10.3389/ pha .2023.1214351
AY.4 a ian was isola ed om a pa ien in Co onou, Benin in July 2021
(GISAID: EPI_ISL_4566935) (Yadoule on e al., 2022). The Omic on
BA.2 a ian was isola ed om a pa ien in Schleswig-Hols ein, Ge many
in Janua y 2022 (GISAID: EPI_ISL_9553926).
S ocks o animal expe imen a ion we e gene a ed on Ve oE6-
TMPRSS2 cells and i a ed on Ve oE6 cells. P io o animal
in ec ion, all i us s ocks we e s o ed a −80°C.
S ocks o in i o expe imen a ion we e bo h gene a ed and
i a ed on Ve oE6 cells. Fo SARS-CoV-2 in ec ion o cell
cul u es, be ween 2 × 10
5
and 3 × 10
5
cells pe mL we e seeded in
6-well pla es o 24-well pla es. A e 24 h, cells we e in ec ed wi h
SARS-CoV-2 in a se um- ee medium. A e 1 h, i us dilu ions we e
emo ed, and he wells we e washed wice wi h PBS and efilled wi h
DMEM (supplemen ed as desc ibed p e iously). Samples we e aken
a he indica ed ime poin s. The ull sequence iden i y o B.1, Del a
AY.4, and Omic on BA.2 SARS-CoV-2 s ocks o in i o expe imen s
was confi med wi h NGS and RT-PCR/Sange sequencing, and can be
made a ailable upon eques . Lineage assignmen was e ified wi h he
FIGURE 7
Epigalloca echin, epigalloca echin galla e, and axi olin inhibi SARS-CoV-2 B.1 p opaga ion dose dependen ly. (A-B) Calu-3 cells we e in ec ed wi h
SARS-CoV-2 (MOI = 0.0005) and ea ed wi h 7 defined low molecula weigh cons i uen s o EPs
®
7630 using 10 μg/mL, as well as DMSO as a ehicle
con ol (A), and addi ionally o cpd C (epigalloca echin), cpd F (epigalloca echin galla e), and cpd G ( axi olin) in a dilu ion se ies o 0.5–10 μg/mL (B).
Vi us-con aining supe na an s we e collec ed 24 h pos -in ec ion and i al i e s we e de e mined as PFU/mL by plaque i a ion assay. Fo bo h
Calu-3 (C) and A549-ACE2 cells (D), compound oxici y was e alua ed by pe o ming a CellTi e Glo assay in a dilu ion ange o 0.5–512 μg/mL o each
compound and 0.5%–5.12% DMSO ( o e alua e ehicle oxici y) a 24 h pos -in ec ion. Da a a e de i ed om n= 3 biological samples. S a is ical
significance is indica ed by (*) as de e mined by wo-way ANOVA o he da a wi h Dunne ’s mul iple compa ison es . As e isks a e shown only o
significan ly di e en da a se s. (*) = p<0.05; (**) = p<0.01; (***) = p<0.001; (****) = p<0.0001.
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