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. Ae iology-speci ic es ima es o he global and egional incidence and
mo ali y o dia hoeal diseases commonly ansmi ed h ough ood. PLoS
One 2015;10(12):e0142927.
[3] Vinje J. Ad ances in labo a o y me hods o de ec ion and yping o no o i us. J
Clin Mic obiol 2015;53(2):373–81.
[4] Blaze ic V, Lappalainen S, Nu minen K, Huh i L, Vesika i T. No o i us VLPs and
o a i us VP6 p o ein as combined accine o childhood gas oen e i is.
Vaccine 2011;29(45):8126–33.
[5] Tamminen K, Lappalainen S, Huh i L, Vesika i T, Blaze ic V. T i alen
combina ion accine induces b oad he e ologous immune esponses o
no o i us and o a i us in mice. PLoS One 2013;8(7):e70409.
[6] Jones MK, Wa anabe M, Zhu S, G a es CL, Keyes LR, G au KR, e al. En e ic
bac e ia p omo e human and mouse no o i us in ec ion o B cells. Science
2014;346(6210):755–9.
[7] E ayebi K, C aw o d SE, Mu akami K, B oughman JR, Ka andika U, Tenge VR,
e al. Replica ion o human no o i uses in s em cell-de i ed human en e oids.
Science 2016;353(6306):1387–93.
[8] Reeck A, Ka anagh O, Es es MK, Opekun AR, Gilge MA, G aham DY, e al.
Se ological co ela e o p o ec ion agains no o i us-induced gas oen e i is. J
In ec Dis 2010;202(8):1212–8.
[9] Ha ing on PR, Lindesmi h L, Youn B, Moe CL, Ba ic RS. Binding o No walk
i us-like pa icles o ABH his o-blood g oup an igens is blocked by an ise a
om in ec ed human olun ee s o expe imen ally accina ed mice. J Vi ol
2002;76(23):12335–43.
[10] Lindesmi h LC, Fe is MT, Mullan CW, Fe ei a J, Debbink K, Swans om J, e al.
B oad blockade an ibody esponses in human olun ee s a e immuniza ion
wi h a mul i alen no o i us VLP candida e accine: immunological analyses
om a phase I clinical ial. PLoS Med 2015;12(3):e1001807.
[11] Uusi-Ke ula H, Tamminen K, Malm M, Vesika i T, Blaze ic V. Compa ison o
human sali a and syn he ic his o-blood g oup an igens usage as ligands in
no o i us-like pa icle binding and blocking assays. Mic obes In ec 2014;16
(6):472–80.
[12] Tacke CO, Sz ein MB, Losonsky GA, Wasse man SS, Es es MK. Humo al,
mucosal, and cellula immune esponses o o al No walk i us-like pa icles in
olun ee s. Clin Immunol 2003;108(3):241–7.
[13] Lindesmi h L, Moe C, Lependu J, F elinge JA, T eano J, Ba ic RS. Cellula and
humo al immuni y ollowing Snow Moun ain i us challenge. J Vi ol 2005;79
(5):2900–9.
[14] Lindesmi h LC, Donaldson E, Leon J, Moe CL, F elinge JA, Johns on RE, e al.
He e o ypic humo al and cellula immune esponses ollowing No walk i us
in ec ion. J Vi ol 2010;84(4):1800–15.
[15] Malm M, Tamminen K, Vesika i T, Blaze ic V. No o i us-speci ic memo y T cell
esponses in adul human dono s. F on Mic obiol 2016;7:1570.
[16] LoBue AD, Lindesmi h LC, Ba ic RS. Iden i ica ion o c oss- eac i e no o i us
CD4+ T cell epi opes. J Vi ol 2010;84(17):8530–8.
[17] Malm M, Tamminen K, Vesika i T, Blaze ic V. Type-speci ic and c oss- eac i e
an ibodies and T cell esponses in no o i us VLP immunized mice a e a ge ed
bo h o conse ed and a iable domains o capsid VP1 p o ein. Mol Immunol
2016;78:27–37.
[18] Huh i L, Blaze ic V, Nu minen K, Koho T, Hy onen VP, Vesika i T. A compa ison
o me hods o pu i ica ion and concen a ion o no o i us GII-4 capsid i us-
like pa icles. A ch Vi ol 2010;155(11):1855–8.
[19] Huh i L, Tamminen K, Vesika i T, Blaze ic V. Cha ac e iza ion and
immunogenici y o no o i us capsid-de i ed i us-like pa icles pu i ied by
anion exchange ch oma og aphy. A ch Vi ol 2013;158(5):933–42.
[20] Tamminen K, Huh i L, Koho T, Lappalainen S, Hy onen VP, Vesika i T, e al. A
compa ison o immunogenici y o no o i us GII-4 i us-like pa icles and P-
pa icles. Immunology 2012;135(1):89–99.
[21] Be s MR, B enchley JM, P ice DA, De Rosa SC, Douek DC, Roede e M, e al.
Sensi i e and iable iden i ica ion o an igen-speci ic CD8+ T cells by a low
cy ome ic assay o deg anula ion. J Immunol Me hods 2003;281(1–
2):65–78.
[22] Malm M, Uusi-Ke ula H, Vesika i T, Blaze ic V. High se um le els o
no o i us geno ype-speci ic blocking an ibodies co ela e wi h p o ec ion
om in ec ion in child en. J In ec Dis 2014;210(11):1755–62.
[23] Malm M, Tamminen K, Lappalainen S, Uusi-Ke ula H, Vesika i T, Blaze ic V.
Geno ype conside a ions o i us-like pa icle-based bi alen no o i us
accine composi ion. Clin Vaccine Immunol 2015;22(6):656–63.
[24] A ma RL, Be ns ein DI, Ha o CD, Al-Ib ahim MS, Chen WH, Fe ei a J, e al.
No o i us accine agains expe imen al human No walk Vi us illness. N Engl J
Med 2011;365(23):2178–87.
[25] Tamminen K, Huh i L, Vesika i T, Blaze ic V. P e-exis ing immuni y o
no o i us GII-4 i us-like pa icles does no impai de no o immune
esponses o no o i us GII-12 geno ype. Vi al Immunol 2013;26(2):167–70.
M. Malm e al. / Vaccine 36 (2018) 484–490 489
[26] Ramani S, A ma RL, Es es MK. Epidemiology o human no o i uses and
upda es on accine de elopmen . Cu Opin Gas oen e ol 2014;30(1):25–33.
[27] Ramani S, Es es MK, A ma RL. Co ela es o p o ec ion agains no o i us
in ec ion and disease-whe e a e we now, whe e do we go? PLoS Pa hog
2016;12(4):e1005334.
[28] LoBue AD, Lindesmi h L, Youn B, Ha ing on PR, Thompson JM, Johns on RE,
e al. Mul i alen no o i us accines induce s ong mucosal and sys emic
blocking an ibodies agains mul iple s ains. Vaccine 2006;24(24):5220–34.
[29] Debbink K, Lindesmi h LC, Donaldson EF, Ba ic RS. No o i us immuni y and
he g ea escape. PLoS Pa hog 2012;8(10):e1002921.
[30] Lambe PH, Liu M, Sieg is CA. Can success ul accines each us how o induce
e icien p o ec i e immune esponses? Na Med 2005;11(4 Suppl):S54–62.
[31] Puschnik A, Lau L, C omwell EA, Balmaseda A, Zompi S, Ha is E. Co ela ion
be ween dengue-speci ic neu alizing an ibodies and se um a idi y in p ima y
and seconda y dengue i us 3 na u al in ec ions in humans. PLoS Negl T op Dis
2013;7(6):e2274.
[32] Delgado MF, Co iello S, Monsal o AC, Melendi GA, He nandez JZ, Ba alle JP,
e al. Lack o an ibody a ini y ma u a ion due o poo Toll-like ecep o
s imula ion leads o enhanced espi a o y syncy ial i us disease. Na Med
2009;15(1):34–41.
[33] Blaze ic V, Malm M, Vesika i T. Induc ion o homologous and c oss- eac i e
GII.4-speci ic blocking an ibodies in child en a e GII.4 New O leans no o i us
in ec ion. J Med Vi ol 2015;87(10):1656–61.
[34] Tomo VT, Osbo ne LC, Dol i DV, Sonnenbe g GF, Mon icelli LA, Mans ield K,
e al. Pe sis en en e ic mu ine no o i us in ec ion is associa ed wi h
unc ionally subop imal i us-speci ic CD8 T cell esponses. J Vi ol 2013;87
(12):7015–31.
[35] Chachu KA, LoBue AD, S ong DW, Ba ic RS, Vi gin HW. Immune mechanisms
esponsible o accina ion agains and clea ance o mucosal and lympha ic
no o i us in ec ion. PLoS Pa hog 2008;4(12):e1000236.
[36] Zhu S, Rege D, Wa anabe M, Hickman D, Moussa che N, Jesus DM, e al.
Iden i ica ion o immune and i al co ela es o no o i us p o ec i e immuni y
h ough compa a i e s udy o in a-clus e no o i us s ains. PLoS Pa hog
2013;9(9):e1003592.
[37] Melhem NM. No o i us accines: co ela es o p o ec ion, challenges and
limi a ions. Hum Vaccin Immuno he 2016;12(7):1653–69.
[38] Wilkinson TM, Li CK, Chui CS, Huang AK, Pe kins M, Liebne JC, e al.
P eexis ing in luenza-speci ic CD4+ T cells co ela e wi h disease p o ec ion
agains in luenza challenge in humans. Na Med 2012;18(2):274–80.
[39] G i in DE, Lin WH, Pan CH. Measles i us, immune con ol, and pe sis ence.
FEMS Mic obiol Re 2012;36(3):649–62.
[40] Blaze ic V, Sahgal N, Kessle HA, Landay AL, Shea e GM. T cell esponses o
ecall an igens, alloan igen, and mi ogen o HIV-in ec ed pa ien s ecei ing
long- e m combined an i e o i al he apy. AIDS Res Hum Re o i uses
2000;16(17):1887–93.
[41] Sede RA, Da ah PA, Roede e M. T-cell quali y in memo y and p o ec ion:
implica ions o accine design. Na Re Immunol 2008;8(4):247–58.
[42] Riddle MS, Walke RI. S a us o accine esea ch and de elopmen o
no o i us. Vaccine 2016;34(26):2895–9.
[43] Lindesmi h LC, Mallo y ML, Jones TA, Richa dson C, Goodwin RR, Baehne F,
e al. Impac o p e-exposu e his o y and hos gene ics on an ibody a idi y
ollowing no o i us accina ion. J In ec Dis 2017;215(6):984–91.
[44] Vigano S, U zschneide DT, Pe eau M, Pan aleo G, Zehn D, Ha a i A. Func ional
a idi y: a measu e o p edic he e icacy o e ec o T cells? Clin De Immunol
2012;2012:153863.
[45] S one JD, Che in AS, K anz DM. T-cell ecep o binding a ini ies and kine ics:
impac on T-cell ac i i y and speci ici y. Immunology 2009;126(2):165–76.
[46] Hanley PJ, Melenho s JJ, Niki o ow S, Scheinbe g P, Blaney JW, Demmle -
Ha ison G, e al. CMV-speci ic T cells gene a ed om nai e T cells ecognize
a ypical epi opes and may be p o ec i e in i o. Sci T ansl Med 2015;7
(285):285 a63.
[47] Zhang SQ, Pa ke P, Ma KY, He C, Shi Q, Cui Z, e al. Di ec measu emen o T
cell ecep o a ini y and sequence om nai e an i i al T cells. Sci T ansl Med
2016;8(341):341 a77.
[48] B en ille VA, Me he ingham RL, Gunn B, Du an LG. High a idi y cy o oxic T
lymphocy es can be selec ed in o he memo y pool bu hey a e exquisi ely
sensi i e o unc ional impai men . PLoS One 2012;7(7):e41112.
[49] Ba y M, Bleackley RC. Cy o oxic T lymphocy es: all oads lead o dea h. Na
Re Immunol 2002;2(6):401–9.
[50] Cal o-Calle JM, S ug I, Nas ke MD, Bake SP, S e n LJ. Human CD4+ T cell
epi opes om accinia i us induced by accina ion o in ec ion. PLoS Pa hog
2007;3(10):1511–29.
[51] Ose o C, Pe e s B, Pasque o V, Mou a si M, Sidney J, Panchana han V, e al.
Dissocia ion be ween epi ope hie a chy and immunop e alence in CD8
esponses o accinia i us wes e n ese e. J Immunol 2008;180
(11):7193–202.
[52] Romani N, Flache V, T ipp CH, Spa be F, Ebne S, S oi zne P. Ta ge ing skin
dend i ic cells o imp o e in ade mal accina ion. Cu Top Mic obiol
Immunol 2012;351:113–38.
[53] Banche eau J, S einman RM. Dend i ic cells and he con ol o immuni y.
Na u e 1998;392(6673):245–52.
[54] Van Damme P, Oos e huis-Ka eja F, Van de Wielen M, Almago Y, Sha on O,
Le in Y. Sa e y and e icacy o a no el mic oneedle de ice o dose spa ing
in ade mal in luenza accina ion in heal hy adul s. Vaccine 2009;27
(3):454–9.
[55] Tamminen K, Malm M, Vesika i T, Blaze ic V. Mucosal An ibodies Induced by
In anasal bu No In amuscula Immuniza ion Block No o i us GII.4 Vi us-
Like Pa icle Recep o Binding. Vi al Immunol 2016;29(5):315–9.
[56] Ramani S, Neill FH, Opekun AR, Gilge MA, G aham DY, Es es MK, e al.
Mucosal and cellula immune esponses o No walk i us. J In ec Dis
2015;212(3):397–405.
[57] Sunda a ajan A, Sangs e MY, F ey S, A ma RL, Chen WH, Fe ei a J, e al.
Robus mucosal-homing an ibody-sec e ing B cell esponses induced by
in amuscula adminis a ion o adju an ed bi alen human no o i us-like
pa icle accine. Vaccine 2015;33(4):568–76.
[58] A ma RL, Be ns ein DI, Lyon GM, T eano JJ, Al-Ib ahim MS, G aham DY, e al.
Se ological Co ela es o P o ec ion agains a GII.4 No o i us. Clin Vaccine
Immunol 2015;22(8):923–9.
490 M. Malm e al. / Vaccine 36 (2018) 484–490