Functionality and avidity of norovirus-specific antibodies and T cells induced by GII.4 virus-like particles alone or co-administered with different genotypes

Func ionali y and a idi y o no o i us-speci ic an ibodies and T cells induced by GII.4 i us-like pa icles alone o co-adminis e ed wi h di e en geno ypes Ma ia Malm, Ki si Tamminen, Su i Heinimäki, Timo Vesika i, Vesna Blaze ic ⇑ Vaccine Resea ch Cen e , Uni e si y o Tampe e, Bioka u 10, 33520 Tampe e, Finland Uni e si y o Tampe e, Facul y o Medicine and Li e Sciences, Tampe e, Finland a icle in o A icle his o y: Recei ed 6 July 2017 Recei ed in e ised o m 10 No embe 2017 Accep ed 4 Decembe 2017 A ailable online 13 Decembe 2017 Keywo ds: No o i us VLP An ibodies T cell immuni y Deg anula ion Deli e y ou e In e e ence abs ac No o i us (NoV) is he main cause o acu e gas oen e i is wo ldwide ac oss all age g oups. Cu en NoV accine candida es a e based on non-in ec ious highly immunogenic i us-like pa icles (VLPs) p oduced in cell cul u es in i o. As NoVs in ec ing human popula ion a e highly di e gen , i is p oposed ha he accine should con ain a leas wo di e en NoV geno ypes, po en ially a ec ing he immunogenici y o each o he . We in es iga ed he immunogenici y o NoV GII.4 VLPs adminis e ed by in amuscula (IM) o in ade mal (ID) injec ions o BALB/c mice ei he alone o co-deli e ed wi h genog oup I (GI) and o he genog oup GII VLPs. Se um NoV-speci ic IgG binding an ibody i e s and an ibody unc ionali y in e ms o a idi y and blocking po en ial we e assessed. Fu he mo e, he speci ici y and unc ional a idi y o CD4 + and CD8 + T cell esponses we e analyzed using syn he ic pep ides p e iously iden i ied o con ain NoV VP1 P2 domain-speci ic H-2 d epi opes. The esul s showed ha IM and ID immuniza ion induced compa able GII.4-speci ic an ibodies and T cell esponses. Simila magni ude and unc ionali y o an i- bodies and in e e on-gamma p oducing T cells we e de eloped using mono alen GII.4 VLPs o di e en geno ype combina ions. Fo he i s ime, deg anula ion assay using mul icolo low cy ome y showed ha NoV GII.4-speci ic CD8 + T cells had cy o oxic T lymphocy e pheno ype. To conclude, ou esul s demons a e ha he e is no immunological in e e ence e en i up o i e di e en NoV VLP geno ypes we e co-adminis e ed a he same ime. Fu he mo e, no inhibi ion o NoV-speci ic an ibody unc ional- i y o he magni ude, speci ici y and a ini y o T cell esponses was obse ed in any o he immunized animals, obse a ions ele an o he de elopmen o a mul i alen NoV VLP accine. Ó2017 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). 1. In oduc ion Acu e gas oen e i is (GE) caused by no o i uses (NoVs) a ec s people o all ages wo ldwide wi h se e e ou comes especially in young child en and he elde ly [1,2]. Highly con agious NoVs a e gene ically di e se wi h con inuously eme ging new a ian s ha leads o as sp eading in human popula ion h ough immune escape. NoVs a e membe s o he Calici i idae amily wi h mo e han 30 ecognized geno ypes in ec ing humans ha belong o genog oups (G) I, II, and IV [3]. The icosahed al i al pa icle o NoV, wi h 38 nm diame e , is composed o 180 copies o majo capsid p o ein, i al p o ein 1 (VP1). NoV i us-like pa icles (VLPs), o med by spon aneous sel -assembly o VP1 in i o, a e mo phologically and an igenically iden ical o he i us. VLPs has con ibu ed signi ican ly o he NoV esea ch, as hey a e success- ully used o s uc u al and immunogenici y s udies, and as p omising NoV accine candida es. Due o he high gene ic a i- abili y o NoVs and a lack o c oss-p o ec i e immuni y be ween GI and GII NoVs, i is belie ed ha a accine should con ain a min- imum o one VLP om each genog oup. Ou g oup has ecen ly p oposed a NoV candida e accine consis ing o GI.3 and GII.4 VLPs in a combina ion wi h o a i us (RV) VP6, o p e en wo majo causes o childhood i al GE wo ldwide [4,5]. Al hough cell cul u e sys ems o g ow h o NoV in i o ha e ecen ly been es ablished, a adi ional neu aliza ion assay is s ill no in use [6,7]. Ins ead, a subse o NoV-speci ic an ibodies ha block binding o NoV VLPs o hei cellula ca bohyd a e ligands, his o-blood g oup an igens (HBGA), a e ega ded as co ela es o p o ec ion om in ec ion [8–11]. The ole o NoV-speci ic T cell esponses in p o ec ion om NoV in ec ion [12–16] has no ye h ps://doi.o g/10.1016/j. accine.2017.12.009 0264-410X/Ó2017 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). ⇑ Co esponding au ho a : Vaccine Resea ch Cen e , Uni e si y o Tampe e, Bioka u 10, FI-33520 Tampe e, Finland. E-mail add ess: [email p o ec ed] (V. Blaze ic). Vaccine 36 (2018) 484–490 Con en s lis s a ailable a ScienceDi ec Vaccine jou nal homepage: www.else ie .com/loca e/ accine been es ablished and equi es u he esea ch. Using ma ix pep- ide pools, we ha e ecen ly iden i ied se en immunodominan H- 2 d es ic ed NoV-speci ic T cell epi opes, including a highly con- se ed uni e sal GII NoV-speci ic CD8 + epi ope (pep ide 99-45, NNYDPTEEIPAPLGTPDF) and GII.4-1999 a ian -speci ic CD4 + T cell epi ope (pep ide 99-50, TRAHKATVSTGSVHFTPK) [17]. In he p esen s udy we in es iga ed NoV GII.4-speci ic an i- body unc ionali y (a idi y and blocking ac i i y) and T cell esponses in mice immunized wi h GII.4-1999 VLPs alone o co- deli e ed wi h di e en VLPs belonging o GI and GII NoVs, wi h he p ima y ocus on mu ual inhibi ion o immunological in e e - ence. In addi ion, NoV-speci ic cy o oxic T lymphocy e (CTL) esponses we e in es iga ed o he i s ime. 2. Ma e ial and me hods 2.1. Recombinan p o eins and syn he ic pep ides Fi e di e en NoV capsid VLPs de i ed om GII.4-1999 ( e e - ence s ain accession no: AF080551), GII.4-2009 New O leans (NO; e e ence s ain accession no: GU445325), GII.12 ( e e ence s ain accession no:AJ277618), GI.1 (accession no: AY502016.1), and GI.3 ( e e ence s ain accession no: AF414403) VLPs we e p o- duced in baculo i us-insec cell sys em and pu i ied by suc ose g adien ul acen i uga ion as desc ibed in de ails ea lie [4,18]. The pu i y, iden i y and mo phology o VLPs we e de e mined by SDS-PAGE, immunoblo ing, and elec on mic oscopy using p e i- ously desc ibed p ocedu es [19]. Se en y- ou syn he ic pep ides ep esen ing he en i e 539 amino acid (aa) sequence o GII.4-1999 NoV VP1 we e syn hesized by Synpep ide Co., L d (Shanghai, China) as 18-me s o e lapping by 11 aa. The pu i y was >70% as de e mined by high-p essu e liq- uid ch oma og aphy. Each lyophilized pep ide was dissol ed in DMSO and u he dilu ed in s e ile PBS o use in he assays. In addi ion, all indi idual pep ides we e pooled (GII.4-99 pool) as ecen ly desc ibed [15]. The GII.4-99 pool was used a a p e- de e mined concen a ion o 2 l g/ml in all assays. Indi idual GII.4 pep ides, 99-45 ( 309 NNYDPTEEIPAPLGTPDF 326 ) and 99-50 ( 344 TRAHKATVSTGSVHFTPK 361 ), a p edic ed [17] mouse H-2 d 9- me epi ope ( 318 PAPLGTPDF 326 , included in he 99-45 sequence), and wo nega i e con ol pep ides, 10-me ( 139 TMFPHIIVDV 148 ) and 17-me (o albumin, OVA 323 ISQAVHAAHAEINEAGR 339 ), we e syn he ized as desc ibed abo e and used a a inal concen a ion o 4 l g/ml. 2.2. Immuniza ions BALB/c (H-2 d ) mice we e ob ained om En igo RMS BV ( o - me ly Ha lan Labo a o ies, Ho s , Ne he lands) and immunized a 7 weeks o age a e a week o acclima iza ion. Fo y mice we e di ided in o 8 g oups (5 mice/g oup) and mice we e immunized ei he in amuscula ly (IM) a he igh caudal high muscle o in ade mally (ID) a he base o he ail (Table 1). The dose o 10 mgo 50 l g o GII.4-1999 VLPs was adminis a ed in a 50 l l ol- ume/dose a week 0 and week 3, a s anda d p ocedu e used by ou labo a o y [4,5]. A g oup o mice ecei ed a mix u e o GII VLPs (GII.4-1999, GII.4 NO and GII.12, 20 mg each) by IM deli e y acco d- ing o he same schedule. Addi ional g oup o mice ecei ed a mix- u e o GII.4 VLPs (as abo e) and a mix u e o GI VLPs (GI.1 and GI.3) simul aneously a he di e en si es. Two nega i e con ol g oups o mice ecei ed a ca ie only (phospha e-bu e ed saline, PBS) ei he IM o ID. No ex e nal adju an s we e used. Two weeks a e he second immuniza ion mice we e sac i iced and se um and spleen cells we e collec ed and p epa ed as p e iously desc ibed [4,5] o he analysis o NoV-speci ic an ibodies and T cell esponses. Mice wel a e was moni o ed h oughou he s udy and expe imen s we e pe o med in acco dance wi h he guideli- nes o he Finnish Na ional Animal Expe imen Boa d. 2.3. Se um IgG an ibody i e and a idi y Se um GII.4-speci ic IgG an ibodies we e de ec ed by enzyme- linked immunoso ben assay (ELISA) as desc ibed elsewhe e in de ail [4,5]. In b ie , wo old dilu ed se um samples we e analyzed on NoV VLP-coa ed pla es. IgG an ibodies we e de ec ed wi h HRP- conjuga ed an i-mouse IgG (Sigma Ald ich, Sain Louis, MO) ol- lowed by o-Phenylenediamine dihyd ochlo ide (OPD)-subs a e (Sigma-Ald ich). The op ical densi y (OD) was measu ed a 490 nm in a mic opla e eade (Vic o 2 1420, Pe kin Elme , Wal ham, MA). The mean end-poin i e s o he indi idual se um we e de e mined as he ecip ocal o he highes se um dilu ion gi ing an OD abo e he se cu -o alue (mean OD o nega i e con ol mice se um wells + 3 SD) and a leas 0.100 OD. A idi y assay was conduc ed as he IgG ELISA assay desc ibed abo e using 1:200 se um dilu ion, bu addi ional wo 8 M u ea incuba ion s eps we e included o emo e low a idi y an ibodies acco ding o p e iously published me hod [20]. A idi y index was calcula ed as (OD wi h u ea/OD wi hou u ea) 100%. 2.4. Blocking assay Blocking assay was used o de ec an ibodies p e en ing NoV GII.4-1999 VLP binding o he syn he ic HBGAs as p e iously desc ibed in de ail [11]. B ie ly, syn he ic bio inyla ed Le d (H ype 1)-PAA-Bio in (Glyco ech, Gai he sbu g, MD) was coa ed on Neu A idin pla es (Pie ce, Rock o d, IL). A e 1 h p e-incuba ion a 37 °C, a mix u e o NoV VLPs (0.4 mg/ml) and se ially wo old dilu ed se um samples we e pla ed and incuba ed o 2 h a 4 °C. The bound VLPs we e de ec ed using NoV VLP ype-speci ic human se um, ollowed by goa an i-human IgG (H + L)-HRP (In i ogen, Ca lsbad, CA) and OPD-subs a e (Sigma-Ald ich). The OD was measu ed as desc ibed abo e. Maximum binding was de e mined by VLP lacking mouse se a. The esul s a e exp essed as he mean blocking index (%) calcula ed as 100% [(OD wells wi h VLP se um mix/OD maximum binding OD) 100%]. Blocking i e 50 (BT50) was exp essed as he ecip ocal o he highes se um dilu- ion blocking 50% o he maximum VLP binding. 2.5. IFN- c ELISPOT assay NoV-speci ic T cell esponses we e de e mined by he abili y o sho syn he ic pep ides o induce ex i o IFN- c p oduc ion by splenocy es o immunized mice using in e e on-gamma (IFN- c ) enzyme-linked immunospo (ELISPOT) assay [5]. Liquid ni ogen Table 1 Expe imen al and con ol g oups o immunized mice. Mice we e immunized in amuscula ly (IM) o in ade mally (ID) a day 0 and 21 wi h he indica ed dose and e mina ed a day 35. G oup Immunogen Dose (mg) Rou e I GII.4-1999 VLP 10 IM II GII.4-1999 VLP 10 ID III GII.4-1999 VLP 50 IM IV GII.4-1999 VLP 50 ID V GII-mix VLPs (GII.4-1999, GII.4 NO, GII.12) 60 (20 each) IM VI GII-mix VLPs (GII.4-1999, GII.4 NO, GII.12)+ GI-mix VLPs (GI.1 and GI.3) 60 + 40 (20 each) IM ID VII PBS – IM VIII PBS – ID PBS, phospha e-bu e ed saline. M. Malm e al. / Vaccine 36 (2018) 484–490 485 ozen splenocy es we e hawed and added o he an i-mouse IFN- c an ibody coa ed Mul isc een HTS-IP il e pla es a 0.1 10 6 cells/well in cul u e medium (CM) con aining 5% FBS (Sigma- Ald ich). The ollowing pep ides we e used o s imula ion: GII.4- 1999 pep ide pool, he wo single pep ides 99-45 and 99-50, a p e- dic ed 9-me H-2 d epi ope, and a nega i e 10-me con ol pep ide. Addi ionally, dose i a ions (0.05–2 mg/ml) o he pep ides 99-45 and 99-50 we e pe o med o de e mine he unc ional a idi y o T cells o he epi opes. CM was used as a backg ound con ol and Concana alin A (10 mg/ml, Sigma-Ald ich) as a cell iabili y con ol. A e 20 h incuba ion a 37 °C in a humidi ied 5% CO 2 a mosphe e , he spo s we e de ec ed by bio inyla ed an i-mouse IFN- c mono- clonal an ibody, ollowed by s ep a idin-ALP and BCIP/NBT sub- s a e (all om Mab ech Ab, Nacka S and, Sweden) acco ding o a p e iously desc ibed p o ocol [5]. The pla es we e analyzed by CTL-Eu ope GmbH (Bonn, Ge many) and he esul s we e exp essed as mean spo o ming cells (SFC) pe 10 6 splenocy es. The expe imen s we e pe o med in duplica es and each expe i- men was epea ed a leas wice. 2.6. Deg anula ion assay Deg anula ion assay was pe o med o de e mine cy o oxic po en ial o he 99-45 pep ide-speci ic CD8 + T mouse splenocy es. The cells (1 10 6 ) we e s imula ed wi h 4 mg/ml 99-45 pep ide in he p esence o 3 mg/ml CD107a (clone 1D4B, BD Biosciences, San Jose, CA) FITC-conjuga ed an ibody. As con ols, uns imula ed cells (spon aneous deg anula ion), nega i e con ol pep ide (OVA) and PMA/ionomycin (Sigma-Ald ich) s imula ed cells we e included. A e 1 h p e-incuba ion a 37 °C and 5% CO 2 , he p o ein anspo inhibi o s b e eldin A and monensin (GolgiPlug and GolgiS op, BD Biosciences) we e added and incuba ion was con inued o e nigh . T ea ed cells we e washed, blocked wi h a an i-mouse CD16/ CD32 (Fc Block, Clone 2.4G2, BD Biosciences) and s ained wi h BD Ho izon Fixable iabili y s ain 780 o li e cells ga ing. Cells we e su ace s ained wi h Pe CP-Cy5.5 luo och ome-conjuga ed CD8-speci ic an ibody, ollowed by ea men wi h BD Cy o ix/ Cy ope m Plus ki (BD Biosciences) and in acellula s aining wi h PE-Cy7 conjuga ed CD3-speci ic an ibody. The cells we e acqui ed on BD FACSCan o II low cy ome e and analyzed using FlowJo .10 so wa e (Th eeS a Inc., San Ca los, CA). CD3 + cells we e ga ed om li e lymphocy e ga e and analyzed o CD8 and CD107a exp ession. 2.7. S a is ics The s a is ical di e ences be ween he expe imen al g oups we e de e mined by he K uskal-Wallis es , Fishe ’s exac es o he Mann-Whi ney U es , as applicable. S a is ical analyses we e pe o med using IBM SPSS S a is ics o Windows (IBM Co p., A monk, NY) e sion 23. S a is ical signi icance was de ined as a p alue o <.05. 3. Resul s 3.1. Simila le els o NoV GII.4-1999 speci ic se um IgG an ibodies induced by IM and ID deli e y o GII.4 VLPs alone o co-deli e ed wi h o he VLP geno ypes Immuniza ion o he mice wi h GII.4 VLPs induced obus geno ype-speci ic IgG an ibodies i espec i e o he dose, deli e y ou e o co-adminis a ion o o he geno ype VLPs a he same ime (Fig. 1A). The GII.4-speci ic se um IgG i e s we e simila in all expe imen al g oups (p > .05), wi h end-poin i e s 102,400 o 204,800 o all VLP immunized mice (Fig. 1B). 3.2. NoV GII.4-1999-speci ic an ibody blocking and a idi y a e no dependen on deli e y ou e o immunogen composi ion Se a o mice immunized wi h GII.4 VLP (10 mg g oups only), GII VLP-mix, o GI VLP + GII VLP mix ia IM o ID ou e we e u he analyzed o an ibody a idi y and blocking ac i i y. All g oups had de eloped high a idi y an ibodies (>50%) wi h no signi ican di e ence be ween he g oups (p = .161) (Fig. 1C). Fu he mo e, simila blocking ac i i y was obse ed o all deli e ies (Fig. 1D). Se a o each expe imen al g oup eached BT50 o 800. The e o e, no immunological in e e ence due o he compe i ion be ween di e en geno ypes was ound in a idi y o blocking ac i i y o GII.4-speci ic an ibodies. 3.3. Magni ude and speci ici y o NoV GII.4-speci ic T cell esponses Syn he ic pep ide pools and single pep ides we e used o com- pa e T cell esponses o mice immunized wi h 10 l g VLPs alone o co-adminis e ed wi h di e en VLP geno ypes. Splenocy es o immunized and con ol mice we e assayed o IFN- c p oduc ion agains di e en pep ide an igens (Fig. 2A). Each expe imen al g oup bu no he con ol mice esponded o a simila le el (p > .05) o all GII.4-1999 de i ed pep ides es ed, while no esponse was induced by he nega i e 10-me con ol pep ide. The GII.4- 99 pep ide pool induced he highes IFN- c esponse compa ed o o he GII.4-speci ic pep ides (p < .05) in all immunized g oups. All es ed NoV-speci ic pep ides, he 18-me pep ides 99-45 and 99-50, p e iously iden i ied o con ain NoV GII.4-speci ic CD8 + and CD4 + T cell-speci ic epi opes [17], and he 9-me p edic ed T cell epi ope [17], induced speci ic SFC in immunized mice, al hough a esponse o 99-45 was somewha highe (p < .05). 3.4. Func ional a idi y o CD8 + and CD4 + T cells As we did no obse e any di e ences in he magni ude o speci ici y o T cell esponses o di e en expe imen al g oups (Fig. 2A), we u he es ed epi ope unc ional a idi y o CD4 + and CD8 + T cells o mice immunized wi h di e en o mula ions, o de ec possible di e ence. The cells we e s imula ed wi h a dose ange (0.01–2 l g/ml, app. 10 5 –10 3 l M) o he single pep ides 99-45 and 99-50 in he IFN- c ELISPOT assays (Fig. 2B and C). No signi ican di e ences in he esponses we e obse ed be ween he g oups (p > .05). 3.5. CD8 + T cells speci ic o he 99-45 pep ide epi ope unde go deg anula ion CD8 + T cells speci ic o he pep ide 99-45 we e u he es ed o po en ial o lyse he NoV in ec ed cells. Flow cy ome y was used o measu e su ace exp ession o he deg anula ion molecule CD107a on CD3 + CD8 + T cells, a ma ke o an e ec o cy o oxic pop- ula ion [21]. App oxima ely 0.8% o 99–45 pep ide–speci ic CD8 + T cells o mice immunized wi h he GII VLP mix (Fig. 3A) and 0.6% o he cells o mice immunized wi h he GI + GII VLP mix (Fig. 3B) exp essed CD107a a he su ace. No 99-45 pep ide-speci ic deg anula ion was seen wi h nega i e con ol mice cells (Fig. 3C) o when 14-me OVA con ol pep ide was used (da a no shown). 4. Discussion A b oadly p o ec i e NoV VLP-based accine should con ain a leas a single ep esen a i e o GI and GII i uses, as he e is e y li le c oss-p o ec i e immuni y be ween he genog oups [5,14,16,22–24]. Concomi an ly, we ha e ecen ly p oposed a i alen combina ion o GII.4 VLPs, GI.3 VLPs and RV VP6 as a can- 486 M. Malm e al. / Vaccine 36 (2018) 484–490 dida e accine agains childhood GE [4,25]. In he p esen s udy, we de e mined an ibodies and T cell immune esponses o GII.4- 1999 VLPs adminis e ed ei he alone o co-adminis e ed wi h di - e en VLP geno ypes in mice, o de e mine possible immunologi- cal in e e ence o inhibi ion o bo h humo al and cellula immune esponses. As he ep esen a i es o GII NoVs, h ee di e - en VLPs we e used, an ances o GII.4-1999, he mo e ecen GII.4 NO (2009) geno ype and an igenically dis an GII.12 geno ype. GII.4 NoVs ha e been dominan in causing spo adic cases and GE ou b eaks o mo e han 20 yea s, wi h newly eme ging a ian s e e y 2–4 yea s [26]. NoV GI.1 and GI.3 VLPs we e chosen as he ep esen a i es o GI NoVs. High se um IgG an ibody i e s gene a ed agains NoV GII.4 we e de ec ed in mice immunized wi h GII.4-1999 VLPs ei he alone o in any combina ion. Fu he mo e, he unc ionali y o hese an ibodies, in e ms o i us neu alizing po en ial and a id- i y, de e mining he s eng h o molecula in e ac ion be ween polyclonal se um an ibodies and an igens, was e y good. This is impo an as blocking an ibodies a e conside ed he mos signi i- can co ela e o p o ec ion om NoV in ec ion and disease iden i- ied so a [8,22,24,27]. High a idi y an ibodies, p oduced as a esul o B cell a idi y ma u a ion, be e ole a e a ia ions wi hin he a ge epi opes [17,23,28,29] and a e impo an o p o ec ion om i al in ec ions [30–32]. NoV-speci ic blocking an ibodies in humans and mice a e highly ype-speci ic [23,28,33] and he e o e he biological signi icance o o he a ms o p o ec i e immuni y, such as c oss- eac i e T cell esponses, should be conside ed besides blocking o ligand in e ac- ion. I has been sugges ed ha p o ec i e immuni y o NoV migh be pa ially dependen on he ac i a ion o T cell immuni y, [14,34–37], howe e , e y li le is known abou he ole o NoV- speci ic T cells in na u al in ec ion [14] and e en less on he ac- cine induced T cell esponses in humans o expe imen al animal models [35,36]. T cell immuni y has a signi ican ole in p o ec ion om o he i uses such as in luenza [38], measles [39], and human immunode iciency i us [40,41]. To he bes o ou knowledge, ou g oup is he only one who has iden i ied NoV-speci ic T cell epi- opes in na u ally exposed humans [15] and BALB/c mice immu- nized wi h he NoV VLPs [17]. Limi ed numbe o GII.4-speci ic T cell epi opes was de ec ed in a he e ogeneous human popula ion, in con as o se en c oss- eac i e T cell epi opes iden i ied in a single inb ed mouse s ain, sugges ing ha T cell immuni y migh signi ican ly imp o e a e NoV VLP accina ion [15,17]. In con as o naï e mice, in humans he accina ion is in lu- enced by he complex p io NoV exposu e his o y, howe e , he e ec is ye unknown [10,42]. Vaccina ion may imp o e he p e- exis ing immuni y in humans by elici ing s ong ecall immune esponses owa ds conse ed epi opes, despi e he wide gene ic in e - and in agenog oup a iabili y o NoVs [10]. Clinical ials in adul s ha e sugges ed ha accina ion induces c oss- p o ec i e memo y immune esponses, such as c oss-blocking an ibodies, di ec ed owa ds s ains encoun e ed in he pas , in Fig. 1. No o i us GII.4-1999-speci ic se um IgG esponses. Indi idual e mina ion se um an ibodies shown as he g oup mean OD alues wi h he s anda d e o s o he mean (A) and end-poin i e s analyzed om he g oup-wise pooled se a (B). Mice we e immunized IM o ID wi h GII.4-1999 VLPs alone (g oups I o IV), GII VLP mix (g oup V), o GI + GII VLP mix (g oup VI). Mice ecei ing ca ie (PBS) only we e used as nega i e con ols (C l, g oups VII and VIII). Ho izon al dashed line (A and B) indica es a posi i i y cu - o (OD > 0.100). GII.4-speci ic IgG mean a idi y indices (%) wi h s anda d e o o he means (C) and pooled se um blocking an ibody i e s (D) in g oups immunized wi h GII.4 VLPs alone (10 mg), GII mix, o GI + GII-mix. Ho izon al dashed line indica es 50% a idi y (C) o blocking (D) index. n.s., s a is ically no signi ican (p > .05). M. Malm e al. / Vaccine 36 (2018) 484–490 487 Fig. 2. NoV GII.4-speci ic T cell esponses. Splenocy es o mice immunized IM o ID wi h 10 mg GII.4 VLP alone, GII VLP mix o GI + GII VLP mix we e analyzed o IFN- c p oduc ion by ELISPOT assay. The con ol mice (C l) ecei ed PBS only. Comple e GII.4-99 pep ide pool, 18-me single pep ides 99-45 and 99-50, and a p edic ed 9-me T cell epi ope we e used o s imula e he cells. An i ele an 10-me pep ide se ed as a nega i e con ol and cul u e media (CM) as a backg ound con ol (A). S imula ion o he cells wi h inc easing concen a ions o he pep ides 99-45 (B) and 99-50 (C) was used o de e mine he unc ional a idi y o T cells. Mean IFN- c spo - o ming cells pe 10 6 splenocy es o epea ed expe imen s wi h s anda d e o s o he mean a e shown. P alue < .05 was conside ed s a is ically signi ican . n.s., s a is ically no signi ican (p > .05). Fig. 3. Exp ession o CD107a in NoV-speci ic CD8 T cells. The g oup-wise pooled splenocy es o mice immunized wi h GII VLP mix (A) o GI + GII VLP mix (B), o splenocy es o he con ol mice (C), we e s imula ed wi h he single pep ide 99-45, in he p esence o CD107a FITC and p o ein anspo inhibi o s, be o e low cy ome y analysis on FACS Can o II. E en s (%) shown a e ga ed o li e CD3 + CD8 + T cells and ci cled a e CD8 + CD107a + cells. 488 M. Malm e al. / Vaccine 36 (2018) 484–490 addi ion o elici ing immune esponses agains s ains included in he accine [10,43]. In his s udy, he T cell esponses we e compa able in all expe - imen al g oups es ed o he CD4 + T cell epi ope 99-50 ( 344 - TRAHKATVSTGSVHFTPK 361 ), CD8 + T cell epi ope 99-45 ( 309 NNYDPTEEIPAPLGTPDF 326 ), and, o he i s ime, o a minimal epi ope ( 318 PAPLGTPDF 326 ) con o ming he p edic ed H-2 d binding mo i [17]. The unc ional a idi y is he ac i a ion h eshold o he T cells, desc ibing how well he cells espond o a gi en concen a- ion o an an igen, as e iewed ex ensi ely by Vigano e al. [44]. Conside ing he majo ole o he high unc ional a idi y o T cell ecep o o a gi en pep ide epi ope in d i ing T cell an i i al ac i - i y [45–48], i is o impo ance ha T cell magni ude, speci ici y and a idi y we e simila in all GII.4 VLP immunized mice, ega d- less o he adminis e ed o mula ion. Impo an ly, he esul s o his s udy a e he i s o sugges he ue CTL po en ial o he CD8 + T cells speci ic o he pep ide 99-45, which con ains he highly conse ed epi ope ac oss GII NoV geno- ypes. Inc eased cell su ace CD107a exp ession in immunized mice cells is associa ed wi h loss o in acellula pe o in, and indi- ca es he abili y o hese cells o lyse NoV in ec ed cells [21]. CTLs a e conside ed as he main e ec o s agains in acellula pa ho- gens, con e ing p o ec ion om he illness in he case ha no all ee i us is neu alized a he po o en y, owing o hei abil- i y o lyse he in ec ed cells p io i us eplica ion and elease o new i ions [49]. Unlike in NoV VLP immunized inb ed mice as desc ibed abo e, highly conse ed CD8 + T cell epi opes in humans emain ye o be iden i ied. Al hough human popula ion is he e o- geneous, i is possible ha some p o ein domains in NoV VLPs a e immunop e alen , i.e. con ain epi opes es ic ed by mul iple MHC alleles [50,51]. IM deli e y is he mos commonly used o accina ing adul s, in an s and small child en. E en hough ID immuniza ion, wi h he abundance o an igen p esen ing cells in he close p oximi y, migh be he mos op imal ou e o deli e ing VLPs [52–54], he esul s o his s udy showed ha IM and ID deli e ies induced sim- ila quan i ies and quali ies o NoV GII.4-speci ic an ibodies and CD4 + and CD8 + T cells. This inding is o a signi ican impo ance conside ing he easiness o IM deli e y o small child en, compa ed o ID deli e y. E en hough he impo ance o IgA and mucosal immuni y in p o ec ion om NoV in ec ion and disease has no been de ini ely es ablished, hey a e likely o play p o ec i e ole in NoV in ec ion ansmi ed ia mucosal ou e [55–57]. In GII.4 challenge s udies, p e-exis ing NoV-speci ic se um IgA and muco- sal IgA we e associa ed wi h dec eased equency o in ec ion and se e e illness [58]. Howe e , in he p esen s udy using pa - en e al ou es o immuniza ion, no IgA esponses we e e alua ed. We belie e ha p o ec ion agains NoV e lec s he sum o a - ious immune esponses, including an ibody and cell-media ed, especially T cell, immuni y. Ou esul s show he po en ial o a mul i alen NoV VLP accine o induce humo al and cell- media ed immune esponses wi hou immunological in e e ence. Acknowledgemen s We g a e ully acknowledge he labo a o y pe sonnel o he Vaccine Resea ch Cen e a he Uni e si y o Tampe e o echnical assis- ance gi en. Funding This esea ch did no ecei e any speci ic g an om unding agencies in he public, comme cial, o no - o -p o i sec o s. Con lic o in e es We decla e no con lic s o in e es . Re e ences [1] Lopman BA, S eele D, Ki kwood CD, Pa asha UD. The as and a ied global bu den o no o i us: p ospec s o p e en ion and con ol. PLoS Med 2016;13 (4):e1001999. [2] Pi es SM, Fische -Walke CL, Lana a CF, De leesschauwe B, Hall AJ, Ki k MD, e al. 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