The conserved 3' UTR-derived small RNA NarS mediates mRNA crossregulation during nitrate respiration.

i uses A icle A Soluble Ve sion o Nipah Vi us Glycop o ein G Deli e ed by Vaccinia Vi us MVA Ac i a es Speci ic CD8 and CD4 T Cells in Mice Geo gia Kalodimou 1,2, S enja Vei 1, Syl ia Jany 1, Ul ich Kalinke 3, Ch is ophe C. B ode 4, Ge d Su e 1,2,* and Asisa Volz 1,2 1Ins i u e o In ec ious Diseases and Zoonoses, LMU Munich, 80539 Munich, Ge many; geo gia.kalodimou@mic o. e med.uni-muenchen.de (G.K.); [email p o ec ed] (S.V.); [email p o ec ed] (S.J.); asisa. olz@mic o. e med.uni-muenchen.de (A.V.) 2Ge man Cen e o In ec ion Resea ch (DZIF), pa ne si e Munich, 80539 Munich, Ge many 3Ins i u e o Expe imen al In ec ion Resea ch, TWINCORE, Cen e o Expe imen al and Clinical In ec ion Resea ch, a join en u e be ween he Helmhol z Cen e o In ec ion Resea ch B aunschweig and he Hanno e Medical School, 30625 Hanno e , Ge many; Kalinke.Ul ich@mh-hanno e .de 4Depa men o Mic obiology and Immunology, Uni o med Se ices Uni e si y o he Heal h Sciences, Be hesda, MD 20814, USA; ch is ophe .b [email p o ec ed] *Co espondence: [email p o ec ed]; Tel.: +49-89-2180-2514 Recei ed: 28 No embe 2019; Accep ed: 20 Decembe 2019; Published: 24 Decembe 2019   Abs ac : Nipah i us (NiV) is an eme ging zoono ic i us ha is ansmi ed by ba s o humans and o pigs, causing se e e espi a o y disease and o en a al encephali is. An ibodies di ec ed agains he NiV-glycop o ein (G) p o ein a e known o play a majo ole in clea ing NiV in ec ion and in p o iding accine-induced p o ec i e immuni y. Mo e ecen ly, T cells ha e been also shown o be in ol ed in eco e y om NiV in ec ion. So a , ela i ely li le is known abou he ole o T cell esponses and he an igenic a ge s o NiV-G ha a e ecognized by CD8 T cells. In his s udy, NiV-G p o ein se ed as he a ge immunogen o ac i a e NiV-speci ic cellula immune esponses. Modi ied Vaccinia i us Anka a (MVA), a sa e y- es ed s ain o accinia i us o p eclinical and clinical accine esea ch, was used o he gene a ion o MVA–NiV-G candida e accines exp essing di e en e sions o ecombinan NiV-G. O e lapping pep ides co e ing he en i e NiV-G p o ein we e used o iden i y majo his ocompa ibili y complex class I/II- es ic ed T cell esponses in ype I in e e on ecep o -de icien (IFNAR − / − ) mice a e accina ion wi h he MVA–NiV-G candida e accines. We ha e iden i ied an H2-b- es ic ed noname pep ide epi ope wi h CD8 T cell an igenici y and a H2-b 15me wi h CD4 T cell an igenici y in he NiV-G p o ein. The iden i ica ion o his epi ope and he a ailabili y o he MVA–NiV-G candida e accines will help o e alua e NiV-G-speci ic immune esponses and he po en ial immune co ela es o accine-media ed p o ec ion in he app op ia e mu ine models o NiV-G in ec ion. O no e, a soluble e sion o NiV-G was ad an ageous in ac i a ing NiV-G-speci ic cellula immune esponses using hese pep ides. Keywo ds: eme ging i uses; accina ion; MVA ec o accines; T cell esponses 1. In oduc ion Nipah i us (NiV) is an eme ging zoono ic pa hogen o global conce n ha was anked ecen ly by he Wo ld Heal h O ganiza ion (WHO) as a high-p io i y pa hogen. NiV is a nega i e-sense, single-s anded RNA i us ha is a membe o he genus Henipa i us ( amily Pa amyxo i idae). NiV was i s iden i ied du ing a la ge ou b eak a ec ing humans and pigs in Malaysia and Singapo e in 1999 [ 1 ]. F om 2001 onwa ds, seasonal ou b eaks a e obse ed almos annually in Bangladesh and spo adically Vi uses 2020,12, 26; doi:10.3390/ 12010026 www.mdpi.com/jou nal/ i uses Vi uses 2020,12, 26 2 o 24 in India [ 2 ]. Recen ou b eaks occu ed in Ke ala in May 2018 and June 2019, he i s ime NiV eme ged in sou he n India [ 2 ]. Two s ains o NiV ha e been iden i ied, Malaysia and Bangladesh s ains, which sha e 91.8% sequence homology [ 3 ]. NiV causes se e e espi a o y disease and encephali is [ 4 – 6 ], wi h a e age case a ali y a es (CFR) o 40–75% [ 7 ]. Mo eo e , long- e m neu ological sequelae and e en elapse o encephali is a e obse ed in many su i o s o in ec ions wi h bo h s ains o NiV [8–10]. The na u al ese oi o NiV and Hend a i us (HeV) a e he ui ba s o he genus P e opus, which a e widely dis ibu ed in Asia, Aus alasia, and pa s o A ica [ 11 , 12 ]. Mo eo e , NiV has a b oad species opism and can cause disease in a many animal species [ 13 – 15 ]. NiV can in ec humans ia se e al ou es, which include he consump ion o ood con amina ed wi h ba sec e ions [ 16 ], ansmission om ampli ica ion hos s such as pigs [ 4 ], and di ec human- o-human ansmission be ween e y close con ac s [ 17 ]. Cu en ly he e a e no licensed ea men s o p e en a i e accines a ailable o use in humans, which make he de elopmen o e ec i e p ophylac ic measu es impe a i e. E idence o da e indica es ha he Henipa i us glycop o ein G is a highly p omising a ge o i us-neu alizing an ibodies o coun e ac in ec ions wi h highly pa hogenic henipa i uses. The G glycop o eins o NiV and HeV sha e 83% amino acid sequence homology and a e ype II ansmemb ane p o eins[ 18 ]. Theglycop o einso bo h i uses bind o hehos cell ecep o seph in-B2and eph in-B3[ 19 – 22 ], which a e highly conse ed ac oss many species [ 23 ]. To da e, he mos p omising he apeu ic app oach agains Henipa i us in ec ion is he one based on he applica ion o he expe imen al human monoclonal an ibody m102.4, which binds he eph in-B2 and eph in-B3 ecep o -binding si e on he glycop o eins o HeV (HeV-G) and NiV (NiV-G) [ 24 ]. The p o ec i e e icacy o m102.4 has been success ully e alua ed in di e en p eclinical models including e e s and A ican g een monkeys (AGM) [25–27]. Cu en ly, se e al accines deli e ing NiV-G ha e been shown o p o ec agains le hal challenge in ec ions in p eclinical es ing. These candida e accines include ecombinan i uses de eloped using accinia i us [ 28 ], cana ypox [ 29 ], esicula s oma i is i us (VSV) [ 30 – 34 ], abies i us [ 35 ], measles i us [ 36 ], and adeno i us pla o ms [ 37 , 38 ]. To da e, he only licensed Henipa i us accine is he equine accine agains HeV, Equi ac ® HeV, which was app o ed o use in ho ses in Aus alia in 2012 [ 39 ]. This accine is a subuni accine, which comp ises he soluble o m o HeV-G (HeVsG) [ 19 , 39 – 41 ]. The subuni HeVsG accine has been shown o p o ec agains NiV in ca s [ 42 ], e e s [ 43 ], and non-human p ima es [ 44 ], bu no in pigs [ 45 ]. The abili y o HeVsG o p o ec agains NiV in ec ion in some animal models wa an s he e alua ion o a accine ha con ains a soluble o m o NiV glycop o ein G. The ole o an ibodies in p o ec i e immuniza ion agains NiV in ec ion has been widely epo ed, howe e he e is limi ed in o ma ion on NiV-speci ic T-cell-media ed immuni y. Consequen ly, i is o in e es o be e unde s and he ole o T cells in accine-induced p o ec ion agains NiV. This, in u n, will aide in he de elopmen o new and imp o ed accine candida es. In his s udy, we in es iga ed ecombinan Modi ied Vaccinia i us Anka a (MVA) o deli e y o NiV-G an igens [ 46 ]. We cons uc ed wo MVA–NiV-G accine candida es o exp ess ull-leng h NiV-G o he soluble o m NiVsG. The candida e accines we e gene ically s able and e icien ly eplica ed in p ima y chicken emb yo ib oblas s, a cell cul u e sys em used o manu ac u ing o MVA accines. Impo an ly, accina ion o mice lacking he in e e on alpha/be a ecep o (IFNAR − / − ) elici ed eadily de ec able NiV-G-speci ic CD8 and CD4 T cells. We iden i ied a po en ial H2-b- es ic ed epi ope in he NiV-G ha s imula ed an igen-speci ic CD8 T cells and a po en ial H2-IAb- es ic ed epi ope ha s imula ed an igen-speci ic CD4 T cells. In e es ingly, in compa ison wi h ull-leng h NiV-G, he soluble an igen NiVsG induced signi ican ly s onge epi ope-speci ic T cell esponses. Ou wo k will be ele an o u u e s udies cha ac e izing NiV-G-speci ic T cell esponses in NiV p eclinical models. 2. Ma e ials and Me hods 2.1. Mice IFNAR − / − mice [ 47 ] backc ossed mo e han 20- old on he C57BL/6 backg ound (we e b ed unde speci ied-pa hogen- ee (SPF) condi ions, housed in isola ed cage uni s (IsoCage, Tecniplas , Vi uses 2020,12, 26 3 o 24 Hohenpeißenbe g, Ge many) and had access o ood and wa e ad libi um. All expe imen s we e app o ed by he Go e nmen o Uppe Ba a ia, Munich Ge many and we e pe o med in compliance wi h he Ge man Animal Wel a e Ac (55.2Ve -2532.Ve _02-17-93, 09.01.2017). 2.2. Cells P ima y chicken emb yo ib oblas s (CEF) we e isola ed om 10-day-old SPF chicken emb yos (VALO, Cuxha en, Ge many) and g own in Minimum Essen ial Medium (MEM) (Sigma-Ald ich, Tau ki chen, Ge many) supplemen ed wi h 10% hea -inac i a ed e al bo ine se um (FBS) (Sigma-Ald ich), 1% Penicillin–S ep omycin (Sigma-Ald ich), and 1% MEM nonessen ial amino acid solu ion (Sigma-Ald ich). Human HeLa cells (ATCC CCL-2) we e main ained in MEM supplemen ed wi h 10% hea -inac i a ed FBS (Sigma-Ald ich) and he abo e an ibio ics. DF-1 cells (ATCC CRL-12203) we e g own in VLE Dulbecco’s Modi ied Eagle’s Medium (DMEM) (Me ck, Da ms ad , Ge many) supplemen ed wi h 10% hea -inac i a ed FBS (Sigma-Ald ich), 1% Penicillin–S ep omycin (Sigma-Ald ich), 1% MEM nonessen ial amino acid solu ion (Sigma-Ald ich), and 1% HEPES solu ion (Sigma-Ald ich). Cells we e main ained a 37 ◦ C in a humidi ied 5% CO2a mosphe e. 2.3. Plasmid Cons uc ions The cDNA ha encoded o he en i e 602 amino acid sequence o he NiV-G p o ein (Nipah i us isola e UMMC1, GenBank accession numbe AY029767.1) was modi ied in silico by in oducing silen codon al e a ions o emo e e mina ion signals o accinia i us ea ly ansc ip ion (TTTTTNT) and G/C nucleo ide uns. Fo cons uc ion o he soluble o m o NiV-G p o ein (NiVsG), he cy oplasmic and ansmemb ane domains we e dele ed and an in e nal leade sequence and amino acid linke sequences we e added as desc ibed p e iously [ 40 , 48 ]. Fo accinia i us (VACV)-speci ic ansc ip ional egula ion, we placed he NiVsG gene sequences unde con ol o he syn he ic VACV ea ly/la e p omo e (PmH5) [ 49 ] and used he s ong na u al ea ly VACV p omo e P g [ 50 – 52 ] o exp ession o NiV-G sequences. The cDNAs encoding o NiV-G o NiVsG including he clea age si es o he es ic ion endonucleases Xhol and Apal we e gene a ed by DNA syn hesis (GeneWiz, Leipzig, Ge many). cDNA sequences we e cloned in o he MVA ans e plasmid pLW-73 [ 53 ], which di ec s inse ion o he e ologous sequences o a si e be ween he open eading ames (ORF) o he essen ial i al genes, MVA069R and MVA070L. 2.4. Gene a ion o Recombinan Vi uses Recombinan MVA i uses exp essing he NiVsG and NiV-G p o eins we e gene a ed as desc ibed p e iously [ 54 , 55 ]. To summa ize, CEF cells a 80–90% con luence we e in ec ed wi h non ecombinan MVA (clonal isola e MVA F6) a a mul iplici y o in ec ion (MOI) o 0.05 and ans ec ed wi h ec o plasmid DNA con aining NiVsG o NiV-G gene sequences using X- emeGENE ™ HP DNA T ans ec ion Reagen (Roche Diagnos ics, Penzbe g, Ge many). A e 48 h incuba ion, cells we e ha es ed, and he ecombinan i uses MVA–NiVsG and MVA–NiV-G we e clonally isola ed by sc eening o co-exp ession o he luo escen p o ein ma ke GFP and plaque passaging se e al imes. Resul an ec o i us isola es we e quali y con olled using s anda d p o ocols [ 54 ]. Polyme ase chain eac ion (PCR) analysis o genomic i al DNA se ed o con i m he gene ic iden i y and genomic s abili y o he MVA ec o i uses. The eplica i e capaci y o he ecombinan MVA–NiV i uses compa ed wi h non ecombinan MVA was es ed by mul i-s ep g ow h expe imen s in CEF and human HeLa cells. To gene a e accine p epa a ions, ecombinan MVA–NiV i uses we e ampli ied in CEF, pu i ied by ul acen i uga ion h ough 36% suc ose cushions, and econs i u ed in 10 mM T is-HCl bu e , pH 9.0, o make s ock p epa a ions [54]. Vi uses 2020,12, 26 4 o 24 2.5. Wes e n Blo Analysis o Recombinan P o eins CEF and HeLa cells we e in ec ed wi h ecombinan MVA–NiVsG and MVA–NiV-G i uses a a MOI o 5. Cells in ec ed wi h MVA (MOI 5) and inocula ion medium alone (mock) we e used as con ols. Cell lysa es we e p epa ed o cul u e supe na an s we e collec ed a a ious ime poin s a e in ec ion and s o ed a − 80 ◦ C. Cell-associa ed and sec e ed p o eins we e esol ed by sodium dodecyl sul a e (SDS)-polyac ylamide (10%) gel elec opho esis (SDS-PAGE), and p o eins we e ans e ed on o ni ocellulose memb anes by we elec oblo ing. To in es iga e he glycosyla ion pa e n, p o eins we e p e ea ed using he P o ein Deglycosyla ion Mix II ki (New England Biolabs, Ipswich, MA, USA) acco ding o he manu ac u e ’s ins uc ions p io o SDS PAGE. Memb anes we e blocked wi h blocking bu e , which consis ed o PBS con aining 5% non- a d ied milk powde (Ca l Ro h, Ka ls uhe, Ge many) and 0.1% / Tween 20 (Sigma-Ald ich) o one hou a oom empe a u e. Memb anes we e hen p obed wi h he p ima y an ibody (polyclonal mouse an i-NiV-G (1:2000) o polyclonal abbi an i-NiV-G (1:5000)) dilu ed in blocking bu e o e nigh a 4 ◦ C. Memb anes we e washed h ee imes wi h PBS con aining 0.1% Tween 20 (PBS/T) and p obed wi h goa an i-mouse IgG conjuga ed o ho se adish pe oxidase (HRP) (1:5000; Agilen Dako, Glos up, Denma k) o goa an i- abbi IgG HRP (1:5000; Cell Signaling Technology, Leiden, The Ne he lands). Memb anes we e washed again wi h PBS/T and we e de eloped using Supe Signal ® Wes Du a Ex ended Du a ion subs a e (The mo Fishe Scien i ic, Planegg, Gemany). Blo s we e isualized using a Mic oChemi 4.2 image (DNR Bio-Imaging Sys ems, Ne e Yamin, Is ael). 2.6. Immuno luo escence Con luen monolaye s o HeLa cells we e in ec ed wi h ecombinan MVA–NiVsG o MVA–NiV-G i uses a MOI 0.05. Con ols included MVA (MOI 0.05) and inocula ion medium alone (mock). A e in ec ion, cells we e incuba ed o 16 h in a 37 ◦ C incuba o . Cells we e ixed wi h 4% pa a o maldehyde (PFA) (Sigma-Ald ich) and on some occasions, pe meabilized wi h 0.5% T i on X-100 (Sigma-Ald ich). Cells we e blocked in blocking bu e con aining 1% bo ine se um albumin (BSA) (Sigma-Ald ich). Cells we e s ained wi h polyclonal abbi an i-NiV-G dilu ed 1:10,000 in PBS con aining 0.5% BSA (PBS/BSA). Cells we e washed wi h PBS and s ained wi h he seconda y an ibody goa an i-mouse IgG Alexa Fluo (AF) 568 (1:1000; The mo Fishe Scien i ic) dilu ed in PBS/BSA bu e . Fluo escence was isualized using he Keyence BZ-X710 mic oscope (Keyence, Osaka, Japan). 2.7. Immuniza ion Expe imen s in Mice Mice we e immunized wi h 10 8 PFU in 50 µ L accine bu e (10 mM T is and 140 mM NaCl, pH 7.4) o ecombinan MVA–NiVsG, ecombinan MVA–NiV-G o MVA o accine bu e as a mock accine con ol ia he in ape i oneal o in amuscula ou es. Mice ecei ed ei he one (p ime) o wo (p ime-boos ) immuniza ions o e a 21 day in e al. Fo T cell analysis, mice we e eu hanized 8 days a e immuniza ion. Blood was collec ed on days 0, 18, and 31. Coagula ed blood was cen i uged a 1300 × g o 5 min in MiniCollec ials (G eine Bio-One, Alphen aan den Rijn, The Ne he lands) in o de o sepa a e se um, which was subsequen ly s o ed a −20 ◦C un il u he use. 2.8. Quan i ica ion o To al An igen-Speci ic IgG An ibodies An igen-speci ic IgG esponses induced by immuniza ion wi h he accine candida es we e analyzed by enzyme-linked immunoso ben assay (ELISA) using pu i ied soluble ecombinan NiV glycop o ein G exp essed in mammalian HEK293 cells. Fla bo om 96-well ELISA pla es (Nunc ™ MaxiSo p ™ Pla es, The mo Scien i ic) we e coa ed wi h 50 ng/well ecombinan p o ein (100 µ L olume) and incuba ed o e nigh a 4 ◦ C. Pla es we e washed h ee imes wi h 200 µ L/well PBS/T. Pla es we e blocked wi h blocking bu e con aining 1% bo ine se um albumin (Sigma-Ald ich) and 0.15 M suc ose (Sigma-Ald ich) in PBS o 1 h a 37 ◦ C. Pla es we e hen washed wi h PBS/T as desc ibed abo e. Se a we e se ially dilu ed in PBS con aining 1% BSA (PBS/BSA) h ee- old down he pla e, s a ing a Vi uses 2020,12, 26 5 o 24 a dilu ion o 1:100 (100 µ L olume/well) and incuba ed o 1 h a 37 ◦ C. A e washing, pla es we e incuba ed wi h 100 µ L/well goa an i-mouse IgG conjuga ed HRP (1:2000; Agilen Dako, Denma k) dilu ed in PBS/BSA o 1 h a 37 ◦ C. Pla es we e hen washed wi h PBS/T as desc ibed ea lie . Then, 100 µ L/well 3,3 0 , 5,5 0 -Te ame hylbenzidine (TMB) Liquid Subs a e Sys em o ELISA (Sigma-Ald ich) was added, and pla es we e incuba ed un il a colo change was obse ed. The eac ion was s opped by he addi ion o 100 µ L/well S op Reagen o TMB Subs a e (450 nm, Sigma-Ald ich). The abso bance was measu ed on an ELISA pla e eade a 450 nm wi h a 620 nm e e ence wa eleng h. To al IgG i e s we e calcula ed om he in lec ion poin o he i a ion cu e as loga i hms o he ecip ocal. 2.9. Gene a ion o Syn he ic Pep ides, Design o Pep ide Pools, and Pep ide P edic ion The p o ein sequence o NiV-G was ob ained om he UniP o da abase (ID: Q9IH62). Using an in silico app oach, we iden i ied 130 indi idual syn he ic pep ides ha spanned he ex e nal domain NiV-G p o ein, s a ing om he hi d amino acid o he N- e minal side o he C e minus (amino acids 73–602). Ou pep ide lib a y consis ed o 15me pep ides ha o e lapped by 11me . All pep ides we e syn hesized by The mo Fishe Scien i ic as c ude ma e ial (<50% pu i y) on a 1–4 mg scale. The wo-dimensional pep ide pool ma ix sys em was used o sc eening [ 56 , 57 ]. B ie ly, pep ides we e o ganized in o wo-dimensional ma ix pep ide pools (H1–H10 and V1–V11) con aining 11–13 pep ides. Fo mapping o po en ial CD8 T cell epi opes in posi i e 15me pep ides, e e y possible sequence o pep ides 8–11me in leng h was de e mined. Theo e ical pep ides we e hen analyzed o binding o he mouse majo his ocompa ibili y complex (MHC) class I allele H2-b using he SYFPEITHI da abase. The pep ides o each amino acid sequence leng h we e syn hesized and es ed. Fo iden i ica ion o CD4 T cell epi opes, MHC class II binding p edic ions we e pe o med on 15me pep ides ound wi hin posi i e pep ide pools iden i ied by IFN- γ Enzyme-Linked Immunospo assay (ELISPOT). Using he MHC Class II Binding, T Cell Epi ope P edic ion esou ce o he Immune Epi ope Da abase (IEDB, h ps://www.iedb.o g/), pep ides es ic ed o mouse MHC class II allele H2-IAb we e analyzed using “IEDB Recommended” p edic ion me hod [ 58 ]. The mos p omising candida es we e hen chosen o u he expe imen al epi ope p edic ion s udies. All pep ides we e dissol ed o a concen a ion o 2 mg/mL in PBS, aliquo ed, and s o ed a −20 ◦C un il use. 2.10. T Cell Analysis 2.10.1. Enzyme-Linked Immunospo assay (ELISPOT) T cell analysis by ELISPOT was pe o med as desc ibed p e iously [ 55 ]. B ie ly, spleens we e collec ed om mice 8 days a e he inal immuniza ion. Single-cell suspensions we e p epa ed by easing spleens h ough a 70 µ m cell s aine (Falcon ® Co ning, Co ning, NY, USA). Red blood cells (RBC) we e emo ed using Red Cell Lysis Bu e (Sigma-Ald ich), and cells we e washed and esuspended in RPMI-10, which consis ed o RPMI-1640 (Sigma-Ald ich) con aining 10% hea -inac i a ed FBS (Sigma-Ald ich) and 1% Penicillin–S ep omycin (Sigma-Ald ich). Fo expe imen s ha equi ed CD4 and CD8 T cell pu i ica ion, splenocy es we e incuba ed wi h mouse CD4 and CD8 Mic oBeads (Mil enyi Bio ec, Be gisch Gladbach, Ge many) and p ocessed by nega i e selec ion using he Quad oMACS Sepa a o (Mil enyi Bio ec). IFN- γ -p oducing cells we e measu ed by IFN- γ ELISPOT assay using he Mouse IFN- γ ELISpo PLUS ki (Mab ech, S ockholm, Sweden) as desc ibed in he manu ac u e ’s p o ocol. In summa y, 2 × 10 5 splenocy es we e seeded on o 96-well la bo om pla es (100 µ L/well) (Sa s ed , Nümb ech , Ge many), and 100 µ L/well pep ide pools, subpools, o indi idual pep ides we e added (each pep ide dilu ed o 2 µ g/mL in RPMI-10). A e mixing, he splenocy e/pep ide mix u es we e ans e ed on o pla es p ecoa ed wi h IFN- γ de ec ion an ibody and incuba ed o 48 h a 37 ◦ C. Nons imula ed cells we e used as a mock con ol, and he posi i e con ols cul u es we e ea ed wi h pho bol my is a e ace a e (PMA) and ionomycin (bo h om Sigma-Ald ich) o accinia i us (VACV)-speci ic CD8 T cell epi ope, B8R 20–27 (TSYKFESV) [ 59 ]. A e incuba ing, pla es we e p ocessed Vi uses 2020,12, 26 6 o 24 as desc ibed in he ki manu ac u e ’s p o ocol (Mab ech). Spo s we e coun ed and analyzed using he Au oma ed ELISPOT pla e eade (A. EL. VIS Eli.Scan and A. EL. VIS ELISPOT Analysis So wa e, Hanno e , Ge many). 2.10.2. In acellula Cy okine S aining and Flow Cy ome y Splenocy es we e dilu ed o 1 × 10 7 cells/mL in RPMI-10, and 100 µ L/well (1 × 10 6 cells) was added on o a 96-well U-bo om pla e. Then, 100 µ L/well pep ide dilu ed o 16 µ g/mL in RPMI-10 was added o gi e a inal pep ide concen a ion o 8 µ g/mL. The VACV CD8 T cell epi ope B8R 20–27 ( inal concen a ion o 8 µ g/mL) was used as a posi i e con ol along wi h PMA (10 ng/mL) plus ionomycin (500 ng/mL). PBS dilu ed in RPMI-10 was used as a mock s imula ed con ol. A e pla ing, cells we e incuba ed o 2 h a 37 ◦ C. Then, 20 µ L/well 10x B e eldin A, a Golgi inhibi o ha was p epa ed by dilu ing 1000X B e eldin A (Biolegend, San Diego, CA, USA) in RMPI-10, was added o gi e a inal dilu ion o 1x B e eldin A. Cells we e hen incuba ed o an addi ional 4 h a 37 ◦ C. A e incuba ing, pla es we e cen i uged (500 g o 3 min), and he supe na an was emo ed. Cells we e washed once wi h 200 µ L/well FACS bu e (MACSQuan Running Bu e wi h 2% FBS). Ex acellula s aining was pe o med wi h he ollowing an ibodies dilu ed in FACS bu e : an i-mouse CD3 phycoe i h in (PE)-Cy7 (clone 17A2, 1:100, Biolegend), an i-mouse CD4 B illian Viole 421 (clone GK1.5, 1:600, Biolegend), an i-mouse CD8 α Alexa Fluo 488 (clone 53-6.8, 1:300, Biolegend), and pu i ied CD16/CD32 (Fc block; clone 93, 1:500, Biolegend). Cells we e s ained in 50 µ L/well o 30 min on ice in he da k, hen washed once wi h 200 µ L/well FACS bu e and hen wice wi h 200 µ L/well PBS. Cells we e hen s ained wi h 100 µ L/well o he ixable dead cell iabili y dye Zombie Aqua (1:800, Biolegend) dilu ed in PBS o 30 min on ice in he da k. Cells we e washed wice wi h 200 µ L/well PBS and ixed wi h 100 µ L/well o Fixa ion Bu e (Biolegend) o 20 min a oom empe a u e in he da k. Cells we e washed wice wi h 200 µ L/well PBS and esuspended in 200 µ L/well FACS bu e . Pla es we e s o ed o e nigh a 4 ◦ C, p o ec ed om ligh . The ollowing day, cells we e pe meabilized by washing h ee imes wi h 200 µ L/well 1 × Pe m Wash bu e , which con ained In acellula S aining Pe meabiliza ion Wash Bu e (10 × ) (Biolegend) dilu ed o 1 × wi h dis illed wa e . Cells we e s ained in acellula ly in 100 µ L/well o an i-mouse IFN- γ (clone XMG1.2, 1:200, Biolegend) plus TNF- α (clone MP6-XT22, 1:200, Biolegend) dilu ed in 1X Pe m Wash bu e o 30 min a oom empe a u e, p o ec ed om ligh . Cells we e hen washed h ee imes wi h 200 µ L/well 1X Pe m Wash and hen esuspended in 300 µ L/well FACS bu e . Samples we e il e ed h ough 50 µ m nylon mesh (Se a P y L d., Hun ingwood, NSW, Aus alia) in o 5 mL ound bo om FACS ubes (Sa s ed ). Single-colo con ols we e p epa ed o each FACS analysis using OneComp eBeads ™ Compensa ion Beads (eBioscience, The mo Fishe Scien i ic) o luo opho e-conjuga ed an ibodies and cells o he iabili y dye Zombie Aqua. Da a acquisi ion was pe o med by MACSQuan VYB Flow Analyse (Mil enyi Bio ec), and da a was analyzed using FlowJo (FlowJo LLC, BD Li e Sciences, Ashland, OR, USA). 2.11. S a is ical Analysis Da a we e analyzed using G aphPad P ism e sion 5.0 (G aphPad So wa e Inc., San Diego, CA, USA) and we e exp essed as mean ± s anda d e o o he mean (SEM). S a is ical analysis was pe o med using he unpai ed, wo- ailed - es o compa e wo g oups and one-way ANOVA o compa e h ee o mo e g oups. The h eshold o s a is ical signi icance was p<0.05. 3. Resul s 3.1. Gene a ion and Cha ac e isa ion o MVA Vec o Vaccines Deli e ing NiV-G An igens To gene a e he ecombinan MVA i uses deli e ing NiV-G an igens, we used he gene om NiV Malaysia (isola e UMMC1, GenBank accession numbe AY029767.1) and gene a ed codon-op imized gene sequences encoding a ull-leng h glycop o ein G (NiV-G) o a soluble ex e nal domain G p o ein (NiVsG). These syn he ic gene sequences we e placed unde he ansc ip ional con ol o he Vi uses 2020,12, 26 7 o 24 accinia i us-speci ic p omo e s P g o PmH5 and in oduced in o he MVA genome by homologous ecombina ion a ge ing he in e genic si e be ween he essen ial MVA genes 069 and 070L. The clonal isola ion o he ecombinan i uses MVA–NiVsG and MVA–NiV-G was acili a ed by he co-p oduc ion o he g een luo escen epo e p o ein (GFP), as p e iously desc ibed [ 54 ]. The inal ecombinan i uses con aining he NiV-G gene sequences (MVA–NiV-G and MVA–NiVsG) we e ob ained a e emo al o he GFP epo e gene by in agenomic homologous ecombina ion (Figu e S1A, ma ke gene dele ion). To e i y he iden i y o he desi ed modi ica ion, we pe o med he s anda d quali y con ol expe imen s as desc ibed p e iously [ 54 ]. PCR analysis o i al genomic DNA con i med he p ope inse ion o he ecombinan gene sequences a he a ge si e in he genome o MVA (Figu e S1B–E) and he gene ic cha ac e is ics and s abili y o he ecombinan MVA i uses. We assessed he g ow h beha io o he ecombinan i uses MVA–NiVsG and MVA–NiV-G in mul i-s ep g ow h analyses in human HeLa cells and p ima y CEF, which a e ou inely used o ampli ica ion o ecombinan MVA in accine manu ac u ing (Figu e S1F). MVA–NiVsG and MVA–NiV-G e icien ly eplica ed in CEF and demons a ed an inc ease o in ec i i y i e s ha was compa able o ha ob ained wi h non ecombinan MVA. Howe e , MVA–NiV-G and MVA–NiVsG did no p oduc i ely g ow in human HeLa cells, con i ming ha hey had e ained he cha ac e is ic eplica ion de iciency o MVA in cells o mammalian o igin. These indings co obo a ed he expec ed MVA pheno ype and con i med ha he ecombinan i uses could be handled unde labo a o y condi ions o biosa e y le el 1. O no e, we o iginally gene a ed ano he ecombinan MVA i us using he syn he ic ea ly–la e p omo e PmH5 o ansc ip ional con ol o ecombinan gene exp ession and p oduc ion o he ull-leng h NiV-G p o ein. Howe e , upon g ow h es ing, his candida e i us ailed o each le els o in ec ious p ogeny in CEF o be eligible o la ge-scale ampli ica ion as needed o accine p oduc ion p ocesses. 3.2. Cha ac e isa ion o Recombinan NiV-G P o eins Ou accine candida es, MVA–NiVsG and MVA–NiV-G, should p oduce ecombinan NiV glycop o ein G in i s ull-leng h o m (NiV-G) and, in pa allel, he soluble o m (NiVsG). NiV-G is a 602 amino acid long p o ein consis ing o an N- e minal in e nal domain, a ansmemb ane domain, and a C- e minal ex e nal domain (Figu e 1A). Fo NiVsG p o ein, an in e nal leade sequence and h ee amino acid linke s ha e eplaced he in e nal and ansmemb ane domains (Figu e 1A), as desc ibed p e iously [ 19 , 48 ]. We assessed he co ec exp ession and s udied he cellula localiza ion o he NiV-G and NiVsG by immuno luo escence mic oscopy o MVA–NiV-in ec ed cells immunolabeled wi h an i-NiV-G an ibody, ollowed by a luo escen ly labelled seconda y an ibody. Cell nuclei we e s ained wi h DAPI (300 nM). As an icipa ed, we obse ed di e en pa e ns o g een luo escence, wi h a ying cellula localiza ions depending on he MVA–NiV cons uc . G een luo escence, speci ic o NiV-G, was iden i ied in pe meabilized and nonpe meabilized cells in ec ed wi h MVA–NiV-G (Figu e 1B), bu no in MVA-in ec ed o mock con ol cells. This da a con i med ha he ecombinan ull-leng h NiV-G p o ein encoded by MVA–NiV-G was indeed ancho ed on he cell su ace. In con as , he NiV-G-speci ic s aining in cells in ec ed wi h MVA–NiVsG i us appea ed o be p edominan ly loca ed wi hin he cells and was eadily de ec ed a e pe meabiliza ion. As an icipa ed, we ailed o de ec NiVsG in conside able amoun s wi hou pe meabiliza ion, con i ming ha NiVsG was no exp essed on he cell su ace (Figu e 1B). To u he in es iga e he syn hesis o NiV-G p o eins a e in ec ion wi h MVA–NiVsG and MVA–NiV-G, espec i ely, o al p o eins om in ec ed CEF and HeLa cell cul u es we e analyzed by Wes e n blo using a NiV-G-speci ic an ibody. To al cell lysa es o cul u e supe na an s ob ained om CEF and HeLa cul u es in ec ed wi h ecombinan MVA i us we e sepa a ed by SDS-PAGE and immunoblo ed. We speci ically de ec ed a p o ein wi h an es ima ed molecula mass o app oxima ely 72–75 kDa in lysa es om CEF cells and HeLa cells in ec ed ei he wi h MVA–NiV-G o MVA–NiVsG (Figu e 1C). In he cell lysa es, he glycop o ein was i s de ec able a 4 h pos -in ec ion. The amoun o p o ein inc eased o e ime, esul ing in a p ominen band a 16 h pos -in ec ion, which was main ained h oughou he 72 h ime cou se. To moni o he elease o ecombinan NiV-G p o ein Vi uses 2020,12, 26 8 o 24 om MVA–NiVsG in ec ed cells, we also analyzed he supe na an s o in ec ed CEF and HeLa cell cul u es. A p o ein wi h a molecula mass o app oxima ely 72–75 kDa sized band was obse ed in supe na an s om MVA–NiVsG-in ec ed cells (Figu e 1C), indica ing ha in ec ed cells we e sec e ing he soluble NiV-G p o ein. Sec e ed NiV-G p o ein was i s obse ed a 16 h pos -in ec ion in he supe na an s o MVA–NiVsG-in ec ed cul u es and became especially p ominen by 24 h pos -in ec ion. The le els o sec e ed NiV-G seemed o decline by 72 h pos -in ec ion. We did no howe e de ec NiV-G p o ein in supe na an s o MVA–NiV-G-in ec ed cells. Vi uses 2020, 12, x FOR PEER REVIEW 8 o 25 Figu e 1. Analysis o ecombinan Nipah i us ull-leng h glycop o ein G (NiV-G) p o ein p oduced by cells in ec ed wi h MVA–NiVsG and MVA–NiV-G. (A) Schema ic diag am o ecombinan NiV ull-leng h glycop o ein G (NiV-G) and NiV soluble glycop o ein G (NiVsG). Colo ed ec angles ep esen indi idual p o ein domains, and b acke ed ex displays he s a and end o amino acid sequences o each domain. (B) Immuno luo escence s aining o cells in ec ed wi h MVA–NiVsG and MVA–NiV-G. HeLa cells we e in ec ed a MOI 0.05 wi h he abo e i uses o 16 h. MVA and mock Figu e 1. Analysis o ecombinan Nipah i us ull-leng h glycop o ein G (NiV-G) p o ein p oduced by cells in ec ed wi h MVA–NiVsG and MVA–NiV-G. ( A ) Schema ic diag am o ecombinan NiV ull-leng h Vi uses 2020,12, 26 9 o 24 glycop o ein G (NiV-G) and NiV soluble glycop o ein G (NiVsG). Colo ed ec angles ep esen indi idual p o ein domains, and b acke ed ex displays he s a and end o amino acid sequences o each domain. ( B ) Immuno luo escence s aining o cells in ec ed wi h MVA–NiVsG and MVA–NiV-G. HeLa cells we e in ec ed a MOI 0.05 wi h he abo e i uses o 16 h. MVA and mock in ec ed HeLa cells we e used as con ols. Fixed pe meabilized and ixed nonpe meabilized cells we e immunos ained wi h abbi polyclonal an ibody o NiV-G and he seconda y an ibody an i- abbi Alexa Fluo 488. Nuclei we e s ained wi h DAPI solu ion. Panel shows ep esen a i e pic u es o ixed/pe meabilized and ixed/nonpe meabilized in ec ed HeLa cells a 40 × magni ica ion. ( C ) Wes e n blo analysis o ecombinan NiV-G p o eins p oduced by chicken emb yo ib oblas s (CEF) and HeLa cells in ec ed wi h MVA–NiVsG and MVA–NiV-G. Lysa es and cul u e supe na an s we e collec ed om cell cul u es in ec ed a MOI 5 wi h he abo e i uses, wild- ype MVA o nonin ec ed con ols (mock). Samples we e collec ed a indica ed hou s pos -in ec ion. Cell lysa es and p o eins we e es ed by immunoblo ing using a NiV-G-speci ic polyclonal mouse an ibody. P o ein bands co esponding o he expec ed molecula weigh s o ecombinan NiV-G and NiVsG p o ein (~65 − 70 kDa) a e indica ed. ( D ) Wes e n Blo analysis o ecombinan p o eins p oduced by DF-1 cells in ec ed wi h MVA–NiVsG and MVA–NiV-G a MOI 5 o 36 h. MVA and mock in ec ed cells we e used as con ols. Cell lysa es and cul u e supe na an s we e incuba ed wi h (+) o wi hou ( − ) enzymes o deglycosyla e p o eins, analyzed by SDS-PAGE, and immunoblo ed wi h a abbi polyclonal an ibody o NiV-G. Be a-ac in was used as a loading con ol o lysa es. Solid black a ow ep esen s glycosyla ed ecombinan NiV-G and NiVsG p o ein (~65–70 kDa), and dashed black a ow ep esen s deglycosyla ed ecombinan NiV-G and NiVsG p o ein (~58 kDa). Blue solid a ow ep esen s be a-ac in (~40 kDa). MVA: modi ied Vaccinia i us Anka a. Since he NiVsG gene encoding sequences we e modi ied o esul in he sec e ed soluble e sion (sG), we pe o med addi ional Wes e n blo expe imen s o de e mine whe he he p o ein s ill main ained glycosyla ion si es compa able o wild- ype NiV-G. Lysa es and supe na an s ob ained om cul u es o DF-1 cells in ec ed wi h MVA–NiVsG we e ea ed wi h enzymes ha deglycosyla e p o eins and analyzed by wes e n blo ing. Enzyme ea men esul ed in a educ ion in he molecula mass o ecombinan NiVsG p o ein om 70–75 kDa o 58–60 kDa (Figu e 1D), ma ching he expec ed size o unmodi ied G p o ein. This sugges ed ha ecombinan NiVsG has e ained he no mal glycosyla ion pa e n o wild- ype NiV-G. 3.3. An ibody Responses in Vaccina ed IFNAR−/−Mice To assess he immunogenici y o he ecombinan MVA–NiV-G/NiVsG candida e accines, we accina ed IFNAR − / − mice wi h 10 8 PFU ia he in amuscula ou e a days 0 and 21. Se um samples we e es ed o NiV-G-binding IgG an ibodies by ELISA 18 days a e he i s immuniza ion (P ime) and 10 days a e he second immuniza ion (P ime-Boos ) (Figu e 2). E en a single applica ion o he MVA–NiV-G accines induced abundan le els o NiV-G-speci ic IgG an ibodies in he mice. A e boos e immuniza ion, all accina ed animals p oduced e en highe le els o ci cula ing NiV-G-speci ic an ibodies, wi h he an ibody i e s inc easing by app oxima ely en- old. Vi uses 2020,12, 26 16 o 24 3.4.4. Iden i ica ion o Po en ial H2-IAb-Res ic ed CD4 T Cell Epi opes o NiV-G Ini ially, we also analyzed pu i ied CD4 T cell cul u es om MVA–NiVsG immunized mice o hei abili y o s imula e IFN- γ by ELISPOT using a wo-dimensional pooled-pep ide ma ix sys em (Figu e 3A, Sec ion 3.4.1). In con as o CD8 T cell-en iched splenocy es, he IFN- γ SFC signals in hese cul u es we e lowe (Figu es 3B and 6A). In o de o de e mine de ini i ely which pep ide pools we e abo e backg ound le els, we selec ed a cu o alue o 20 IFN- γ SFC/10 6 cells (Figu e 6A, g ey line). Mean IFN- γ SFC alues abo e backg ound we e obse ed in 8 ou o he 21 pools (pools H3, H4, V2, V3, V5, V6, V9, and V11) (Figu e 6A). Vi uses 2020, 12, x FOR PEER REVIEW 18 o 25 Figu e 6. Ac i a ion o an igen-speci ic CD4 T cells a e immuniza ion wi h he ecombinan MVA candida e accines exp essing NiV-G o NiV-sG. G oups o IFNAR−/− mice (n = 2–7) we e immunized wice wi h MVA–NiVsG, MVA–NiV-G, MVA, o saline (mock) ia he i.p. o i.m. ou e o e a 21 day pe iod. Eigh days a e he inal immuniza ion, CD4 T cell-en iched splenocy es o o al splenocy es we e es imula ed and measu ed by IFN-γ ELISPOT assay o IFN-γ in acellula cy okine s aining (ICS) plus FACS analysis. (A) Sc eening o H2-IAb- es ic ed T cell epi opes o NiV-G in CD4 T cell- en iched splenocy es ob ained om i.p. immunized mice. T cell esponses we e measu ed by IFN-γ ELISPOT assay. G aph shows IFN-γ SFC o CD4 T cell-en iched splenocy es s imula ed wi h pep ide Figu e 6. Ac i a ion o an igen-speci ic CD4 T cells a e immuniza ion wi h he ecombinan MVA candida e accines exp essing NiV-G o NiV-sG. G oups o IFNAR − / − mice (n=2–7) we e immunized Vi uses 2020,12, 26 17 o 24 wice wi h MVA–NiVsG, MVA–NiV-G, MVA, o saline (mock) ia he i.p. o i.m. ou e o e a 21 day pe iod. Eigh days a e he inal immuniza ion, CD4 T cell-en iched splenocy es o o al splenocy es we e es imula ed and measu ed by IFN- γ ELISPOT assay o IFN- γ in acellula cy okine s aining (ICS) plus FACS analysis. ( A ) Sc eening o H2-IAb- es ic ed T cell epi opes o NiV-G in CD4 T cell-en iched splenocy es ob ained om i.p. immunized mice. T cell esponses we e measu ed by IFN- γ ELISPOT assay. G aph shows IFN- γ SFC o CD4 T cell-en iched splenocy es s imula ed wi h pep ide pools H1–H10 and V1–V11. The g ey dashed line ep esen s he g oup mean cu o alue (20 IFN- γ SFC/10 6 cells) o iden i ying posi i e pep ide pools. ( B,C ) Iden i ica ion o H2-IAb- es ic ed candida e epi opes o NiV-G by IFN- γ ICS and FACS analysis. To al splenocy es om i.m. immunized mice we e es imula ed wi h wo p omising candida e H2-IAb- es ic ed 15me pep ides, #49 (LFMTNVWTPPNPNTV) and #50 (NVWTPPNPNTVYHCS), iden i ied by pep ide pool sc eening and in silico H2-IAb-binding p edic ions using he IEDB da abase. ( B ) Rep esen a i e low cy ome y do plo s showing IFN- γ p oduc ion in he splenic CD4 T cell compa men . ( C ) F equency and absolu e numbe (pe 10 6 splenocy es) o IFN- γ +CD4 T cells. Dashed line on g aphs ep esen he cu o o de ini i ely posi i e samples. Di e ences be ween indi idual g oups we e analyzed by one-way ANOVA and Tukey pos -hoc es . As e isks ep esen s a is ically signi ican di e ences be ween wo g oups o a speci ic pep ide. * p<0.05, ** p<0.01. In o de o iden i y po en ial CD4 T cell epi opes o H2-IAb, we pe o med a compu a ional analysis o he 15me pep ides in he i e mos posi i e pep ide pools measu ed by ELISPOT assay (pools H3, H4, V3, V6, and V9). Using MHC II binding p edic ions ob ained om he IEDB online esou ce, we ound wo p omising pep ides. These pep ides we e #49 (LFMTNVWTPPNPNTV) and #50 (NVWTPPNPNTVYHCS) (Table 2). Nex , we used IFN- γ ICS o iden i y di ec ly an igen-speci ic CD4 T cells a e s imula ion wi h hese pep ides. Fo his, splenocy es om mice ha had been accina ed wi h MVA–NiV-G o MVA–NiVsG we e s imula ed wi h pep ides #49 and #50 and analyzed by low cy ome y. A small popula ion o pep ide-speci ic IFN- γ +CD4 T cells was obse ed in he MVA–NiVsG and MVA–NiV-G g oups (Figu e 6B). Due o low equencies o IFN- γ -p oducing cells, we chose a cu o alue o 0.1% o di e en ia e be ween posi i e signals and backg ound. O e all, CD4 T cells om he MVA–NiVsG g oup had a equency o IFN- γ +abo e he cu o ela i e o MVA–NiV-G g oup (Figu e 6C). The mean pe cen age o IFN- γ +CD4 T cells o he wo pep ides was 0.09–0.17% and 0.04–0.07% o MVA–NiVsG and MVA–NiV-G, espec i ely. Mo eo e , he equency and absolu e numbe o IFN- γ -p oducing CD4 T cells in pep ide #50 s imula ed cul u es was signi ican ly highe in he MVA–NiVsG g oup when compa ed wi h he MVA–NiV-G g oup (mean =290 ± 65 and 140 ±46 cells/106 splenocy es espec i ely). Fo pep ide #49, MVA–NiVsG accina ed mice showed a signi ican ly ele a ed equency o IFN- γ +CD4 T cells only. In conclusion, ou da a indica e ha pep ide #50 (NVWTPPNPNTVYHCS) is a p omising H2-IAb- es ic ed CD4 T cell epi ope candida e o NiV-G. The alignmen o he pep ide o he ull amino acid sequence o wild- ype NiV-G is shown in Figu e S3. 4. Discussion The con inuous h ea o suddenly eme ging NiV ou b eaks, pa icula ly in Bangladesh and India, demons a e he need o coun e measu e app oaches eady o use in an immedia e public heal h esponse. A p esen , he e a e no licensed NiV accines o use in humans a ailable. The exis ence o a NiV candida e accine should signi ican ly educe he isk o in ec ion and ansmission o he i us in he case o an ou b eak scena io. The e a e some expe imen al NiV accines ha ha e al eady been es ed in di e en p eclinical animal models. The majo ocus o hese app oaches was o e alua e he immunogenici y and e icacy in he con ex o NiV challenge in ec ion. In hose s udies, e icacy has been mos ly associa ed wi h he gene a ion o NiV-speci ic an ibodies, and immune moni o ing is mainly elying on he de ec ion o i us-neu alizing an ibodies [ 31 , 60 , 61 ]. Howe e , he e is ela i ely li le known abou he induc ion and he ele ance o NiV-speci ic cellula immune esponses. In ha con ex , he a ailabili y o app op ia e ools o in es iga e he ole o T cells in NiV-speci ic immuni y is an impo an p e equisi e in he de elopmen o new accines and he apeu ics. Thus, i will be Vi uses 2020,12, 26 18 o 24 indispensable o moni o in animal models he con ibu ion o i us-speci ic T cells o p o ec i e immuni y bu also o po en ial NiV an igen-speci ic immune pa hology. He e, we iden i ied a majo his ocompa ibili y complex (MHC) haplo ype H2-b- es ic ed pep ide epi ope in he NiV-G p o ein by s imula ing T cells om MVA − NiVsG accina ed IFNAR − / − mice wi h a wo-dimensional (2D) ma ix pool o o e lapping pep ides. IFNAR − / − mice ha e been al eady es ablished as a aluable p eclinical animal model o NiV in ec ion wi h a LD 50 o 8 × 10 3 p u a e in ape i oneal challenge in ec ion [ 15 ]. Mo eo e , in p e ious s udies, we ha e al eady success ully demons a ed ha in e e on ype I ecep o knockou mice (IFNAR − / − ) [ 47 ] can be eadily used as animal models o s udy he immunogenici y and p o ec i e capaci y o MVA immuniza ion [ 62 , 63 ]. He e, we wished o speci ically assess he abili y o MVA-deli e ed NiV-G an igen o induce he ac i a ion o cellula immune esponses in mice. In gene al, he en elope G p o ein is known as he well-conse ed a achmen glycop o ein o bo h HeV and NiV. In a p e ious s udy, a ecombinan adeno-associa ed i us accine exp essing a ull-leng h NiV-G p o ein p o ec ed hams e s in an NiV in ec ion model [ 38 ]. In ano he app oach, a soluble e sion o HeV-G has been enginee ed and showed an e en mo e ad an ageous e icacy when es ed in di e en p eclinical animal models [ 42 , 43 , 64 ]. Using HeVsG, a monoclonal an ibody m102.4 was de i ed and has al eady been success ully es ed as a he apeu ic app oach in humans. To u he enhance Henipa i us G p o ein-induced immunogenici y, we also designed and es ed a soluble e sion o he NiV-G p o ein (NiVsG) simila o he HeVsG an igen used o he gene a ion o m102.4. To compa a i ely e alua e he immunogenici y o he NiVsG p o ein, we also gene a ed an MVA exp essing ull-leng h G. The ecombinan i uses MVA–NiV-G and MVA–NiVsG p oduced s able amoun s o NiV-G an igen upon in i o in ec ion o human cells, indica ing he unimpai ed exp ession o he a ge gene unde ansc ip ional con ol o he syn he ic accinia i us-speci ic ea ly–la e p omo e PmH5 o he s ong na u al ea ly p omo e P g . In he case o MVA–NiVsG, emo al o he ansmemb ane domain and cy oplasmic ail esul ed in he sec e ion o he NiV-G om MVA-in ec ed cells and accumula ion also in he supe na an o cell cul u es, as demons a ed in Wes e n blo analysis and immunos aining. A simila esul was ob ained wi h HeVsG, as exp essed by con en ional ecombinan VACV [ 41 ]. In con as , he ull-leng h MVA-p oduced NiV-G p o ein was no eleased in he supe na an , indica ing he s able p esen a ion on he cell su ace h ough he ansmemb ane domains. Ano he impo an aspec o p ope p o ein exp ession, olding and con o ma ional s abili y is in luenced by he N-glycans. Recen s udies indica ed ha NiV-G N-glycans educe usion e iciency because emo al o some N-glycans caused cell–cell hype usogenici y and inc eased i al en y [ 65 ]. Glycosidase ea men o ull-leng h MVA-p oduced NiV-G esul ed in a polypep ide o 58 kDa, co esponding o he molecula mass p edic ed om he NiV G gene encoding sequences. The glycosidase ea men o he NiVsG also indica ed he p esence o all he N-glycans si es wi hin he soluble e sion o he glycop o ein. A i s in i o e alua ion in mice e ealed ha ea men wi h he MVA–NiV-G and MVA–NiVsG candida e accines esul ed in he induc ion o simila le els o G-binding se um an ibodies, con i ming he immunogenici y o bo h an igen e sions [ 66 ]. In ha con ex , he p esence o binding an ibodies seems o play a subs an ial ole in he blocking o NiV en y, since he mechanism o NiV neu aliza ion is complex and in ol es mo e an igenic si es han hose equi ed o simple ecep o binding [ 67 ]. Howe e , mo e ecen s udies in di e en animal models sugges ha cellula immune esponses a e also in ol ed in media ing p o ec ion agains NiV in ec ion [ 38 , 68 ]. This obse a ion is u he suppo ed by s udies in a pig model o NiV in ec ion showing subs an ial ac i a ion o CD8 T cells a e o al in ec ion wi h NiV [ 45 ]. In line wi h hese p eclinical da a, humans su i ing NiV in ec ion [ 2 ] showed signi ican le els o p oli e a ing (Ki-67+) CD8 T cells, indica ing he p esence o acu e e ec o cells. These da a emphasize ha in addi ion o he humo al immune esponses, T cells could be associa ed wi h eco e y om NiV in ec ion. In a mo e ecen s udy, S oh and cowo ke s con i med he ac i a ion o NiV-speci ic CD8 T cells in mice a e accina ion wi h NiV-like pa icles. These da a u he highligh ha T cells may play a c i ical ole in NiV in ec ion [ 68 ]. Ano he hypo hesis is ha NiV-speci ic T cells could be in ol ed in po en ial immunopa hologies. In his con ex , da a om in ec ions wi h o he neu o opic i uses, Vi uses 2020,12, 26 19 o 24 o example, Wes Nile i us, demons a ed ha an igen-speci ic T cells can open he blood b ain ba ie and con ibu e o i us in ec ions o he b ain [ 69 – 73 ]. Thus, o allow o mo e de ailed s udies cha ac e izing T cells in NiV-associa ed immuni y o pa hogenesis, i is essen ial o iden i y NiV-G pep ide epi opes allowing o he speci ic MHC- es ic ed an igen p esen a ion and he ac i a ion o NiV-speci ic T cells. We iden i ied a noname epi ope NiV-G-9.3 75–83 (RSTDNQAVI). Analysis o his pep ide sequence showed ha NiV-G 9.3 75–83 could be unc ionally conse ed in Hend a i us, bu no Ceda i us (ano he ecognized Henipa i us), G an igens (Figu e S3). S uc u al and unc ional analyses e eal p omiscuous and species-speci ic use o eph in ecep o s by Ceda i us [ 74 ]. This po en ial epi ope will suppo a mo e de ailed expe imen al cha ac e iza ion o T cells induced by NiV in ec ion in he mouse model and hei con ibu ion o pa hogenesis and p o ec ion. In his s udy, we ound ha MVA–NiVsG accina ion induced signi ican ly highe amoun s o NiV-G epi ope-speci ic CD8 T cells compa ed wi h he MVA–NiV-G candida e accine. This was a somewha su p ising obse a ion as he immuniza ions wi h bo h an igens had elici ed e y compa able le els o G-speci ic an ibodies. I is emp ing o specula e ha NiVsG, as a soluble an igen, can igge enhanced T cell esponses because i is a ailable o wo di e en pa hways o an igen p esen a ion. On he one hand, NiVsG as in acellula ly syn he ized an igen is endogenously p ocessed and p esen ed ia MHC-I on he cell su ace o ac i a e CD8 T cells. In addi ion, he NiVsG is sec e ed in high amoun s om he MVA–NiVsG-in ec ed cells, and such ex acellula an igen can e icien ly uel he c oss-p esen a ion pa hway and he eby induce ele a ed CD8 and CD4 T cell immune esponses [ 75 , 76 ]. In e es ingly, he MVA–NiVsG candida e accine also p o ed o apidly induce NiV-G epi ope-speci ic CD8 T cells a e single-dose applica ion. These da a a e o ele ance, since he epidemiology o he mo e ecen NiV ou b eaks demons a ed ha a po en ial accine candida e should apidly p o ec . Impo an ly, an H2b- es ic ed epi ope has been iden i ied in IFNAR − / − mice, which se e as an es ablished small animal model o NiV in ec ion [ 15 ]. In addi ion, we showed he induc ion o NiV-G-speci ic CD4 T cells upon p ime-boos immuniza ion in he IFNAR − / − wi h he MVA–NiV accines and using pep ides o in i o s imula ion, as iden i ied by using MHC II binding p edic ions ob ained om he IEDB online esou ce (www.iedb.o g). This da a goes well along wi h he hypo hesis ha CD4 T cell esponses a e also signi ican ly ele a ed upon in ec ion [ 77 ]. Again, MVA–NiVsG accina ion esul s in mo e e icien ac i a ion o CD4 T cell esponses. Fu he expe imen s will be needed o cha ac e ize he con ibu ion o NiV G-speci ic CD4 T cells o NiV in ec ion in mo e de ail. Taken oge he , ou indings showed he ac i a ion o NiV-G-speci ic T cells in IFNAR − / − mice ollowing accina ion wi h MVA-based candida e accines. We con i med he iden i ica ion o po en ial H2-b- es ic ed NiV-G CD8 and CD4 T cell pep ide epi opes. In his s udy we also demons a ed ha an MVA–NiVsG candida e accine may ha e supe io immunogenici y, esul ing in NiV-speci ic an ibodies and T cells in IFNAR − / − mice. These da a emphasize he p omise o u u e s udies in his animal model u he e alua ing he ole o NiV-speci ic T cells ac i a ed by he G-9.3 75–83 and G-50 269–283 pep ide epi opes, in bo h accine-induced p o ec ion and po en ial con ibu ion o NiV-induced pa hologies. Supplemen a y Ma e ials: The ollowing a e a ailable online a h p://www.mdpi.com/1999-4915/12/1/26/s1, Figu e S1: Gene a ion and cha ac e iza ion o ecombinan MVA–NiVsG and MVA–NiV-G, Figu e S2: IFN- γ p oduc ion by CD8 and CD4 T cells s imula ed by he NiV-G pep ide 9.3, Figu e S3: Amino acid alignmen o H2-b- and H2-IAb- es ic ed pep ides, Figu e S4: Quali y o ac i a ed CD8 T cells om mice immunized wi h ecombinan MVA exp essing NiV-G. Au ho Con ibu ions: G.K., S.V., C.C.B., G.S. and A.V. concei ed and designed he expe imen s; G.K., S.V., S.J. and A.V. pe o med he expe imen s; G.K., S.V., U.K., C.C.B., G.S. and A.V. analyzed he da a; G.K., G.S. and A.V. w o e he pape . All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This wo k was suppo ed by he Fede al Minis y o Educa ion and Resea ch (BMBF), g an numbe DZIF TTU 01.805. Acknowledgmen s: U sula Klos e meie , Pa izia Bone , Johannes Dö ing and Axel G oß p o ided aluable help wi h he animal s udies. Vi uses 2020,12, 26 20 o 24 Con lic s o In e es : The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , o in he decision o publish he esul s. Re e ences 1. Chua, K.B.; Bellini, W.J.; Ro a, P.A.; Ha cou , B.H.; Tamin, A.; Lam, S.K.; Ksiazek, T.G.; Rollin, P.E.; Zaki, S.R.; Shieh, W.-J.; e al. Nipah Vi us: A Recen ly Eme gen Deadly Pa amyxo i us. Science 2000 ,288, 1432–1435. [C ossRe ] [PubMed] 2. A unkuma , G.; De adiga, S.; McEl oy, A.K.; P abhu, S.; Sheik, S.; Abdulmajeed, J.; Robin, S.; Sushama, A.; Jaya am, A.; Ni u , S.; e al. Adap i e Immune Responses in Humans Du ing Nipah Vi us Acu e and Con alescen Phases o In ec ion. Clin. In ec . Dis. 2019,69, 1752–1756. [C ossRe ] [PubMed] 3. Ha cou , B.H.; Lowe, L.; Tamin, A.; Liu, X.; Bankamp, B.; Bowden, N.; Rollin, P.E.; Come , J.A.; Ksiazek, T.G.; Hossain, M.J.; e al. Gene ic cha ac e iza ion o Nipah i us, Bangladesh, 2004. Eme g. In ec . Dis. 2005 ,11, 1594–1597. [C ossRe ] [PubMed] 4. Ang, B.S.P.; Lim, T.C.C.; Wang, L. Nipah Vi us In ec ion. J. Clin. Mic obiol. 2018 ,56, e01875-17. [C ossRe ] [PubMed] 5. Dawes, B.E.; F eibe g, A.N. Henipa i us in ec ion o he cen al ne ous sys em. Pa hog. Dis. 2019 , 77. [C ossRe ] 6. Hossain, M.J.; Gu ley, E.S.; Mon gome y, J.M.; Bell, M.; Ca oll, D.S.; Hsu, V.P.; Fo men y, P.; C oisie , A.; Be he a , E.; Faiz, M.A.; e al. Clinical P esen a ion o Nipah Vi us In ec ion in Bangladesh. Clin. In ec . Dis. 2008,46, 977–984. [C ossRe ] 7. Sha ma, V.; Kaushik, S.; Kuma , R.; Yada , J.P.; Kaushik, S. Eme ging ends o Nipah i us: A e iew. Re . Med. Vi ol. 2019,29, e2010. [C ossRe ] 8. Sej a , J.J.; Hossain, J.; Saha, S.K.; Gu ley, E.S.; Banu, S.; Hamadani, J.D.; Faiz, M.A.; Siddiqui, F.M.; Mohammad, Q.D.; Mollah, A.H.; e al. Long- e m neu ological and unc ional ou come in Nipah i us in ec ion. Ann. Neu ol. 2007,62, 235–242. [C ossRe ] 9. Tan, C.T.; Goh, K.J.; Wong, K.T.; Sa ji, S.A.; Chua, K.B.; Chew, N.K.; Mu ugasu, P.; Loh, Y.L.; Chong, H.T.; Tan, K.S.; e al. Relapsed and la e-onse Nipah encephali is. Ann. Neu ol. 2002,51, 703–708. [C ossRe ] 10. Ng, B.Y.; Lim, C.C.; Yeoh, A.; Lee, W.L. Neu opsychia ic sequelae o Nipah i us encephali is. J. Neu opsychia y Clin. Neu osci. 2004,16, 500–504. [C ossRe ] 11. Clay on, B.A.; Wang, L.F.; Ma sh, G.A. Henipa i uses: An Upda ed Re iew Focusing on he P e opid Rese oi and Fea u es o T ansmission. Zoonoses Public Heal h 2013,60, 69–83. [C ossRe ] [PubMed] 12. Kessle , M.K.; Becke , D.J.; Peel, A.J.; Jus ice, N.V.; Lunn, T.; C owley, D.E.; Jones, D.N.; Eby, P.; S á nchez, C.A.; Plow igh , R.K. Changing esou ce landscapes and spillo e o henipa i uses. Ann. N. Y. Acad. Sci. 2018 , 1429, 78–99. [C ossRe ] 13. Wong, K.T.; Ong, K.C. Pa hology o acu e henipa i us in ec ion in humans and animals. Pa hol. Res. In . 2011,2011, 567248. [C ossRe ] [PubMed] 14. Geisbe , T.W.; Feldmann, H.; B ode , C.C. Animal challenge models o henipa i us in ec ion and pa hogenesis. Cu . Top. Mic obiol. Immunol. 2012,359, 153–177. [C ossRe ] [PubMed] 15. Dhond , K.P.; Ho a , B. Henipa i us in ec ions: Lessons om animal models. Pa hogens 2013 ,2, 264–287. [C ossRe ] 16. Rahman, M.A.; Hossain, M.J.; Sul ana, S.; Homai a, N.; Khan, S.U.; Rahman, M.; Gu ley, E.S.; Rollin, P.E.; Lo, M.K.; Come , J.A.; e al. Da e palm sap linked o Nipah i us ou b eak in Bangladesh, 2008. Vec o Bo ne Zoono ic Dis. 2012,12, 65–72. [C ossRe ] 17. Nikolay, B.; Salje, H.; Hossain, M.J.; Khan, A.K.M.D.; Sazzad, H.M.S.; Rahman, M.; Daszak, P.; S öhe , U.; Pulliam, J.R.C.; Kilpa ick, A.M.; e al. T ansmission o Nipah Vi us—14 Yea s o In es iga ions in Bangladesh. N. Engl. J. Med. 2019,380, 1804–1814. [C ossRe ] 18. Khe awa , D.; B ode , C.C. A Func ional Henipa i us En elope Glycop o ein Pseudo yped Len i i us Assay Sys em. Vi ol. J. 2010,7, 312. [C ossRe ] 19. Bonapa e, M.I.; Dimi o , A.S.; Bossa , K.N.; C ame i, G.; Mungall, B.A.; Bishop, K.A.; Choudh y, V.; Dimi o , D.S.; Wang, L.-F.; Ea on, B.T.; e al. Eph in-B2 ligand is a unc ional ecep o o Hend a i us and Nipah i us. P oc. Na l. Acad. Sci. USA 2005,102, 10652–10657. [C ossRe ] Vi uses 2020,12, 26 21 o 24 20. Neg e e, O.A.; Le oney, E.L.; Aguila , H.C.; Be olo i-Cia le , A.; Naza ian, R.; Tajya , S.; Lee, B. Eph inB2 is he en y ecep o o Nipah i us, an eme gen deadly pa amyxo i us. Na u e 2005 ,436, 401–405. [C ossRe ] 21. Neg e e, O.A.; Wol , M.C.; Aguila , H.C.; En e lein, S.; Wang, W.; Mühlbe ge , E.; Su, S.V.; Be olo i-Cia le , A.; Flick, R.; Lee, B. Two Key Residues in Eph inB3 A e C i ical o I s Use as an Al e na i e Recep o o Nipah Vi us. PLoS Pa hog. 2006,2, e7. [C ossRe ] [PubMed] 22. Pa ch, J.R.; C ame i, G.; Wang, L.F.; Ea on, B.T.; B ode , C.C. Quan i a i e analysis o Nipah i us p o eins eleased as i us-like pa icles e eals cen al ole o he ma ix p o ein. Vi ol. J. 2007 ,4, 1. [C ossRe ] [PubMed] 23. Bossa , K.N.; Tachedjian, M.; McEache n, J.A.; C ame i, G.; Zhu, Z.; Dimi o , D.S.; B ode , C.C.; Wang, L.-F. Func ional s udies o hos -speci ic eph in-B ligands as Henipa i us ecep o s. Vi ology 2008 ,372, 357–371. [C ossRe ] [PubMed] 24. Zhu, Z.; Dimi o , A.S.; Bossa , K.N.; C ame i, G.; Bishop, K.A.; Choudh y, V.; Mungall, B.A.; Feng, Y.-R.; Choudha y, A.; Zhang, M.-Y.; e al. Po en Neu aliza ion o Hend a and Nipah Vi uses by Human Monoclonal An ibodies. J. Vi ol. 2006,80, 891–899. [C ossRe ] 25. Geisbe , T.W.; Mi e, C.E.; Geisbe , J.B.; Chan, Y.-P.; Agans, K.N.; Feldmann, F.; Fen on, K.A.; Zhu, Z.; Dimi o , D.S.; Sco , D.P.; e al. The apeu ic T ea men o Nipah Vi us In ec ion in Nonhuman P ima es wi h a Neu alizing Human Monoclonal An ibody. Sci. T ansl. Med. 2014,6, 242 a82. [C ossRe ] 26. Mi e, C.E.; Sa e ield, B.A.; Geisbe , J.B.; Agans, K.N.; Bo ise ich, V.; Yan, L.; Chan, Y.-P.; C oss, R.W.; Fen on, K.A.; B ode , C.C.; e al. Pa hogenic Di e ences be ween Nipah Vi us Bangladesh and Malaysia S ains in P ima es: Implica ions o An ibody The apy. Sci. Rep. 2016,6, 30916. [C ossRe ] 27. Bossa , K.N.; Zhu, Z.; Middle on, D.; Klippel, J.; C ame i, G.; Bingham, J.; McEache n, J.A.; G een, D.; Hancock, T.J.; Chan, Y.-P.; e al. A Neu alizing Human Monoclonal An ibody P o ec s agains Le hal Disease in a New Fe e Model o Acu e Nipah Vi us In ec ion. PLoS Pa hog. 2009,5, e1000642. [C ossRe ] 28. Guillaume, V.; Con amin, H.; Lo h, P.; Geo ges-Cou bo , M.-C.; Le eu e, A.; Ma ianneau, P.; Chua, K.B.; Lam, S.K.; Buckland, R.; Deubel, V.; e al. Nipah Vi us: Vaccina ion and Passi e P o ec ion S udies in a Hams e Model. J. Vi ol. 2004,78, 834–840. [C ossRe ] 29. Weinga l, H.M.; Be hane, Y.; Caswell, J.L.; Loosmo e, S.; Audonne , J.-C.; Ro h, J.A.; Czub, M. Recombinan Nipah Vi us Vaccines P o ec Pigs agains Challenge. J. Vi ol. 2006,80, 7929–7938. [C ossRe ] 30. Mi e, C.E.; Ve s eeg, K.M.; C oss, R.W.; Agans, K.N.; Fen on, K.A.; Whi , M.A.; Geisbe , T.W. Single injec ion ecombinan esicula s oma i is i us accines p o ec e e s agains le hal Nipah i us disease. Vi ol. J. 2013,10, 353. [C ossRe ] 31. DeBuyssche , B.L.; Sco , D.; Ma zi, A.; P esco , J.; Feldmann, H. Single-dose li e-a enua ed Nipah i us accines con e comple e p o ec ion by elici ing an ibodies di ec ed agains su ace glycop o eins. Vaccine 2014,32, 2637–2644. [C ossRe ] [PubMed] 32. Lo, M.K.; Bi d, B.H.; Cha opadhyay, A.; D ew, C.P.; Ma in, B.E.; Coleman, J.D.; Rose, J.K.; Nichol, S.T.; Spi opoulou, C.F. Single-dose eplica ion-de ec i e VSV-based Nipah i us accines p o ide p o ec ion om le hal challenge in Sy ian hams e s. An i i . Res. 2014,101, 26–29. [C ossRe ] [PubMed] 33. P esco , J.; De Buyssche , B.L.; Feldmann, F.; Ga dne , D.J.; Haddock, E.; Ma ella o, C.; Sco , D.; Feldmann, H. Single-dose li e-a enua ed esicula s oma i is i us-based accine p o ec s A ican g een monkeys om Nipah i us disease. Vaccine 2015,33, 2823–2829. [C ossRe ] [PubMed] 34. De Buyssche , B.L.; Sco , D.; Thomas, T.; Feldmann, H.; P esco , J. Pe i-exposu e p o ec ion agains Nipah i us disease using a single-dose ecombinan esicula s oma i is i us-based accine. NPJ Vaccines 2016 ,1, 16002. [C ossRe ] [PubMed] 35. Keshwa a, R.; Shiels, T.; Pos niko a, E.; Ku up, D.; Wi blich, C.; Johnson, R.F.; Schnell, M.J. Rabies-based accine induces po en immune esponses agains Nipah i us. NPJ Vaccines 2019 ,4, 15. [C ossRe ] [PubMed] 36. Yoneda, M.; Geo ges-Cou bo , M.-C.; Ikeda, F.; Ishii, M.; Naga a, N.; Jacquo , F.; Raoul, H.; Sa o, H.; Kai, C. Recombinan Measles Vi us Vaccine Exp essing he Nipah Vi us Glycop o ein P o ec s agains Le hal Nipah Vi us Challenge. PLoS ONE 2013,8, e58414. [C ossRe ] 37. an Do emalen, N.; Lambe, T.; Sebas ian, S.; Bushmake , T.; Fische , R.; Feldmann, F.; Haddock, E.; Le ko, M.; A anza o, V.A.; Rissanen, I.; e al. A single-dose ChAdOx1- ec o ed accine p o ides comple e p o ec ion agains Nipah Bangladesh and Malaysia in Sy ian golden hams e s. PLoS Negl. T op. Dis. 2019 ,13, e0007462. [C ossRe ] Vi uses 2020,12, 26 22 o 24 38. Ploquin, A.; Sz é csi, J.; Ma hieu, C.; Guillaume, V.; Ba a eau, V.; Ong, K.C.; Wong, K.T.; Cosse , F.-L.; Ho a , B.; Sal e i, A. P o ec ion Agains Henipa i us In ec ion by Use o Recombinan Adeno-Associa ed Vi us–Vec o Vaccines. J. In ec . Dis. 2013,207, 469–478. [C ossRe ] 39. B ode , C.C.; Wei , D.L.; Reid, P.A. Hend a i us and Nipah i us animal accines. Vaccine 2016 ,34, 3525–3534. [C ossRe ] 40. Bossa , K.N.; C ame i, G.; Dimi o , A.S.; Mungall, B.A.; Feng, Y.-R.; Pa ch, J.R.; Choudha y, A.; Wang, L.-F.; Ea on, B.T.; B ode , C.C. Recep o binding, usion inhibi ion, and induc ion o c oss- eac i e neu alizing an ibodies by a soluble G glycop o ein o Hend a i us. J. Vi ol. 2005,79, 6690–6702. [C ossRe ] 41. Colg a e, M.L.; Snelling, H.J.; Shiell, B.J.; Feng, Y.-R.; Chan, Y.-P.; Bossa , K.N.; Xu, K.; Nikolo , D.B.; B ode , C.C.; Michalski, W.P. Si e occupancy and glycan composi ional analysis o wo soluble ecombinan o ms o he a achmen glycop o ein o Hend a i us. Glycobiology 2011 ,22, 572–584. [C ossRe ] [PubMed] 42. McEache n, J.A.; Bingham, J.; C ame i, G.; G een, D.J.; Hancock, T.J.; Middle on, D.; Feng, Y.-R.; B ode , C.C.; Wang, L.-F.; Bossa , K.N. A ecombinan subuni accine o mula ion p o ec s agains le hal Nipah i us challenge in ca s. Vaccine 2008,26, 3842–3852. [C ossRe ] [PubMed] 43. Pallis e , J.A.; Klein, R.; A kins all, R.; Haining, J.; Long, F.; Whi e, J.R.; Payne, J.; Feng, Y.-R.; Wang, L.-F.; B ode , C.C.; e al. Vaccina ion o e e s wi h a ecombinan G glycop o ein subuni accine p o ides p o ec ion agains Nipah i us disease o o e 12 mon hs. Vi ol. J. 2013,10, 237. [C ossRe ] [PubMed] 44. Bossa , K.N.; Rockx, B.; Feldmann, F.; B ining, D.; Sco , D.; LaCasse, R.; Geisbe , J.B.; Feng, Y.R.; Chan, Y.P.; Hickey, A.C.; e al. A Hend a i us G glycop o ein subuni accine p o ec s A ican g een monkeys om Nipah i us challenge. Sci. T ansl. Med. 2012,4, 146 a107. [C ossRe ] [PubMed] 45. Picke ing, B.S.; Ha dham, J.M.; Smi h, G.; Weinga l, E.T.; Dominowski, P.J.; Foss, D.L.; Mwangi, D.; B ode , C.C.; Ro h, J.A.; Weinga l, H.M. P o ec ion agains henipa i uses in swine equi es bo h, cell-media ed and humo al immune esponse. Vaccine 2016,34, 4777–4786. [C ossRe ] [PubMed] 46. Volz, A.; Su e , G. Chap e Fi e-Modi ied Vaccinia Vi us Anka a: His o y, Value in Basic Resea ch, and Cu en Pe spec i es o Vaccine De elopmen . In Ad ances in Vi us Resea ch; Kielian, M., Me enlei e , T.C., Roossinck, M.J., Eds.; Academic P ess: Camb idge, MA, USA, 2017; Volume 97, pp. 187–243. 47. Mulle , U.; S einho , U.; Reis, L.F.; Hemmi, S.; Pa lo ic, J.; Zinke nagel, R.M.; Ague , M. Func ional ole o ype I and ype II in e e ons in an i i al de ense. Science 1994,264, 1918–1921. [C ossRe ] 48. Chan, Y.-P.; Lu, M.; Du a, S.; Yan, L.; Ba , J.; Flo a, M.; Feng, Y.-R.; Xu, K.; Nikolo , D.B.; Wang, L.-F.; e al. Biochemical, Con o ma ional, and Immunogenic Analysis o Soluble T ime ic Fo ms o Henipa i us Fusion Glycop o eins. J. Vi ol. 2012,86, 11457–11471. [C ossRe ] 49. Wya , L.S.; Sho s, S.T.; Mu phy, B.R.; Moss, B. De elopmen o a eplica ion-de icien ecombinan accinia i us accine e ec i e agains pa ain luenza i us 3 in ec ion in an animal model. Vaccine 1996 ,14, 1451–1458. [C ossRe ] 50. Assa sson, E.; G eenbaum, J.A.; Sunds öm, M.; Scha e , L.; Hammond, J.A.; Pasque o, V.; Ose o , C.; Hend ickson, R.C.; Le kowi z, E.J.; Tscha ke, D.C.; e al. Kine ic analysis o a comple e pox i us ansc ip ome e eals an immedia e-ea ly class o genes. P oc. Na l. Acad. Sci. USA 2008,105, 2140–2145. [C ossRe ] 51. Yang, Z.; B uno, D.P.; Ma ens, C.A.; Po cella, S.F.; Moss, B. Simul aneous high- esolu ion analysis o accinia i us and hos cell ansc ip omes by deep RNA sequencing. P oc. Na l. Acad. Sci. USA 2010 ,107, 11513–11518. [C ossRe ] 52. Ma , L.; Lül , A.-T.; F eudens ein, A.; Su e , G.; Volz, A. My is oyla ion inc eases he CD8+T-cell esponse o a GFP p o o ype an igen deli e ed by modi ied accinia i us Anka a. J. Gen. Vi ol. 2016 ,97, 934–940. [C ossRe ] 53. Wya , L.S.; Ea l, P.L.; Xiao, W.; Ame ico, J.L.; Co e , C.A.; Vog , J.; Moss, B. Elucida ing and Minimizing he Loss by Recombinan Vaccinia Vi us o Human Immunode iciency Vi us Gene Exp ession Resul ing om Spon aneous Mu a ions and Posi i e Selec ion. J. Vi ol. 2009,83, 7176–7184. [C ossRe ] [PubMed] 54. K eme , M.; Volz, A.; K eij z, J.H.C.M.; Fux, R.; Lehmann, M.H.; Su e , G. Easy and E icien P o ocols o Wo king wi h Recombinan Vaccinia Vi us MVA. In Vaccinia Vi us and Pox i ology: Me hods and P o ocol; Isaacs, S.N., Ed.; Humana P ess: To owa, NJ, USA, 2012; pp. 59–92. [C ossRe ] 55. Vei , S.; Jany, S.; Fux, R.; Su e , G.; Volz, A. CD8+T Cells Responding o he Middle Eas Respi a o y Synd ome Co ona i us Nucleocapsid P o ein Deli e ed by Vaccinia Vi us MVA in Mice. Vi uses 2018 ,10, 718. [C ossRe ] [PubMed] Vi uses 2020,12, 26 23 o 24 56. Fio e-Ga land, A.; Manso, B.A.; F ied ich, D.P.; Gab iel, E.E.; Finak, G.; Moodie, Z.; He z, T.; De Rosa, S.C.; F ahm, N.; Gilbe , P.B.; e al. Pooled-Pep ide Epi ope Mapping S a egies A e E icien and Highly Sensi i e: An E alua ion o Me hods o Iden i ying Human T Cell Epi ope Speci ici ies in La ge-Scale HIV Vaccine E icacy T ials. PLoS ONE 2016,11, e0147812. [C ossRe ] 57. Malm, M.; Tamminen, K.; Vesika i, T.; Blaze ic, V. No o i us-Speci ic Memo y T Cell Responses in Adul Human Dono s. F on . Mic obiol. 2016,7, 1570. [C ossRe ] 58. Dhanda, S.K.; Mahajan, S.; Paul, S.; Yan, Z.; Kim, H.; Jespe sen, M.C.; Ju z, V.; And ea a, M.; G eenbaum, J.A.; Ma ca ili, P.; e al. IEDB-AR: Immune epi ope da abase—Analysis esou ce in 2019. Nucleic Acids Res. 2019 , 47, W502–W506. [C ossRe ] 59. Tscha ke, D.C.; Ka upiah, G.; Zhou, J.; Palmo e, T.; I ine, K.R.; Hae y a , S.M.; Williams, S.; Sidney, J.; Se e, A.; Bennink, J.R.; e al. Iden i ica ion o pox i us CD8+T cell de e minan s o enable a ional design and cha ac e iza ion o smallpox accines. J. Exp. Med. 2005,201, 95–104. [C ossRe ] [PubMed] 60. Walpi a, P.; Cong, Y.; Jah ling, P.B.; Rojas, O.; Pos niko a, E.; Yu, S.; Johns, L.; Holb ook, M.R. A VLP-based accine p o ides comple e p o ec ion agains Nipah i us challenge ollowing mul iple-dose o single-dose accina ion schedules in a hams e model. NPJ Vaccines 2017,2, 21. [C ossRe ] 61. Middle on, D.; Pallis e , J.; Klein, R.; Feng, Y.-R.; Haining, J.; A kins all, R.; F aze , L.; Huang, J.-A.; Edwa ds, N.; Wa eing, M.; e al. Hend a i us accine, a one heal h app oach o p o ec ing ho se, human, and en i onmen al heal h. Eme g. In ec . Dis. 2014,20, 372–379. [C ossRe ] 62. Volz, A.; Langenmaye , M.; Jany, S.; Kalinke, U.; Su e , G. Rapid Expansion o CD8+T Cells in Wild-Type and Type I In e e on Recep o -De icien Mice Co ela es wi h P o ec ion a e Low-Dose Eme gency Immuniza ion wi h Modi ied Vaccinia Vi us Anka a. J. Vi ol. 2014,88, 10946–10957. [C ossRe ] 63. K eme , M.; Sueze , Y.; Volz, A.; F enz, T.; Majzoub, M.; Hanschmann, K.-M.; Lehmann, M.H.; Kalinke, U.; Su e , G. C i ical Role o Pe o in-dependen CD8+T Cell Immuni y o Rapid P o ec i e Vaccina ion in a Mu ine Model o Human Smallpox. PLoS Pa hog. 2012,8, e1002557. [C ossRe ] [PubMed] 64. Pallis e , J.; Middle on, D.; Wang, L.F.; Klein, R.; Haining, J.; Robinson, R.; Yamada, M.; Whi e, J.; Payne, J.; Feng, Y.R.; e al. A ecombinan Hend a i us G glycop o ein-based subuni accine p o ec s e e s om le hal Hend a i us challenge. Vaccine 2011,29, 5623–5630. [C ossRe ] [PubMed] 65. Bie ing, S.B.; Huang, A.; Vu, A.T.; Robinson, L.R.; B adel-T e heway, B.; Choi, E.; Lee, B.; Aguila , H.C. N-Glycans on he Nipah Vi us A achmen Glycop o ein Modula e Fusion and Vi al En y as They P o ec agains An ibody Neu aliza ion. J. Vi ol. 2012,86, 11991–12002. [C ossRe ] [PubMed] 66. Hayman, D.T.S.; Wang, L.-F.; Ba , J.; Bake , K.S.; Suu-I e, R.; B ode , C.C.; Cunningham, A.A.; Wood, J.L.N. An ibodies o Henipa i us o Henipa-Like Vi uses in Domes ic Pigs in Ghana, Wes A ica. PLoS ONE 2011 , 6, e25256. [C ossRe ] 67. Bossa , K.N.; McEache n, J.A.; Hickey, A.C.; Choudh y, V.; Dimi o , D.S.; Ea on, B.T.; Wang, L.F. Neu aliza ion assays o di e en ial henipa i us se ology using Bio-Plex p o ein a ay sys ems. J. Vi ol. Me hods 2007,142, 29–40. [C ossRe ] [PubMed] 68. S oh, E.; Fische , K.; Schwaige , T.; Saue he ing, L.; F anzke, K.; Maisne , A.; G oschup, M.H.; Blohm, U.; Diede ich, S. Henipa i us-like pa icles induce a CD8 T cell esponse in C57BL/6 mice. Ve . Mic obiol. 2019 , 237, 108405. [C ossRe ] 69. Mus a á , Y.M.; Meu en, L.M.; Coelho, S.V.A.; de A uda, L.B. Pa hways Exploi ed by Fla i i uses o Coun e ac he Blood-B ain Ba ie and In ade he Cen al Ne ous Sys em. F on . Mic obiol. 2019 ,10, 525. [C ossRe ] 70. Wang, T.; Town, T.; Alexopoulou, L.; Ande son, J.F.; Fik ig, E.; Fla ell, R.A. Toll-like ecep o 3 media es Wes Nile i us en y in o he b ain causing le hal encephali is. Na . Med. 2004,10, 1366–1373. [C ossRe ] 71. Roe, K.; Kuma , M.; Lum, S.; O illo, B.; Ne u ka , V.R.; Ve ma, S. Wes Nile i us-induced dis up ion o he blood–b ain ba ie in mice is cha ac e ized by he deg ada ion o he junc ional complex p o eins and inc ease in mul iple ma ix me allop o einases. J. Gen. Vi ol. 2012,93, 1193–1203. [C ossRe ] 72. Diamond, M.S.; Klein, R.S. Wes Nile i us: C ossing he blood-b ain ba ie . Na . Med. 2004 ,10, 1294–1295. [C ossRe ] 73. Wang, Y.; Lobigs, M.; Lee, E.; Mullbache , A. CD8+T cells media e eco e y and immunopa hology in Wes Nile i us encephali is. J. Vi ol. 2003,77, 13323–13334. [C ossRe ] [PubMed] Vi uses 2020,12, 26 24 o 24 74. Laing, E.D.; Na a a na ajah, C.K.; Cheliou Da Sil a, S.; Pe zing, S.R.; Xu, Y.; S e ling, S.L.; Ma sh, G.A.; Wang, L.F.; Amaya, M.; Nikolo , D.B.; e al. S uc u al and unc ional analyses e eal p omiscuous and species speci ic use o eph in ecep o s by Ceda i us. P oc. Na l. Acad. Sci. USA 2019 ,116, 20707–20715. [C ossRe ] [PubMed] 75. Gas eige , G.; Kas enmulle , W.; Ljapoci, R.; Su e , G.; D exle , I. C oss-P iming o Cy o oxic T Cells Dic a es An igen Requisi es o Modi ied Vaccinia Vi us Anka a Vec o Vaccines. J. Vi ol. 2007 ,81, 11925–11936. [C ossRe ] [PubMed] 76. Bu gdo , S.; Kau z, A.; Böhne , V.; Knolle, P.A.; Ku s, C. Dis inc Pa hways o An igen Up ake and In acellula Rou ing in CD4 and CD8 T Cell Ac i a ion. Science 2007,316, 612–616. [C ossRe ] [PubMed] 77. Halpin, K.; Hya , A.D.; Foga y, R.; Middle on, D.; Bingham, J.; Eps ein, J.H.; Rahman, S.A.; Hughes, T.; Smi h, C.; Field, H.E.; e al. P e opid Ba s a e Con i med as he Rese oi Hos s o Henipa i uses: A Comp ehensi e Expe imen al S udy o Vi us T ansmission. Am. J. T op. Med. Hyg. 2011 ,85, 946–951. [C ossRe ] © 2019 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).