In vitro and in vivo effects of Pelargonium sidoides DC. root extract EPs® 7630 and selected constituents against SARS-CoV-2 B.1, Delta AY.4/AY.117 and Omicron BA.2

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. 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