micb-07-01570 Sep embe 29, 2016 Time: 16:25 # 1
ORIGINAL RESEARCH
published: 03 Oc obe 2016
doi: 10.3389/ micb.2016.01570
Edi ed by:
Akihide Ryo,
Yokohama Ci y Uni e si y, Japan
Re iewed by:
Do ian McIl oy,
Uni e si y o Nan es, F ance
Takamasa Ueno,
Kumamo o Uni e si y, Japan
Hideaki Shimizu,
Kawasaki Ci y Ins i u e o Public
Heal h, Japan
*Co espondence:
Vesna Blaze ic
[email p o ec ed]
†These au ho s ha e sha ed i s
au ho ship.
Special y sec ion:
This a icle was submi ed o
Vi ology,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 29 June 2016
Accep ed: 20 Sep embe 2016
Published: 03 Oc obe 2016
Ci a ion:
Malm M, Tamminen K, Vesika i T
and Blaze ic V (2016)
No o i us-Speci ic Memo y T Cell
Responses in Adul Human Dono s.
F on . Mic obiol. 7:1570.
doi: 10.3389/ micb.2016.01570
No o i us-Speci ic Memo y T Cell
Responses in Adul Human Dono s
Ma ia Malm†, Ki si Tamminen†, Timo Vesika i and Vesna Blaze ic*
Vaccine Resea ch Cen e , Uni e si y o Tampe e, Tampe e, Finland
No o i us (NoV) is a leading cause o acu e gas oen e i is in people o all ages
wo ldwide. NoV-speci ic se um an ibodies which block he binding o NoV i us-like
pa icles (VLPs) o he cell ecep o s ha e been ho oughly in es iga ed. In con as ,
only a ew publica ions a e a ailable on he NoV capsid VP1 p o ein-speci ic T cell
esponses in humans na u ally in ec ed wi h he i us. F eshly isola ed pe iphe al blood
mononuclea cells o eigh heal hy adul human dono s p e iously exposed o NoV
we e s imula ed wi h pu i ied VLPs de i ed om NoV GII.4-1999, GII.4-2012 (Sydney),
and GI.3, and IFN-γp oduc ion was measu ed by an ELISPOT assay. In addi ion,
76 o e lapping syn he ic pep ides spanning he en i e 539-amino acid sequence o
GII.4 VP1 we e pooled in o wo-dimensional ma ices and used o iden i y pu a i e
T cell epi opes. Se en o he eigh subjec s p oduced IFN-γin esponse o he
pep ides and i e subjec s p oduced IFN-γin esponse o he VLPs o he same
o igin. In gene al, s onge T cell esponses we e induced wi h he pep ides in each
dono compa ed o he VLPs. A CD8+T cell epi ope in he shell domain o he VP1
(134SPSQVTMFPHIIVDVRQL151) was iden i ied in wo subjec s, bo h ha ing human
leukocy e an igen (HLA)-A∗02:01 allele. To ou knowledge, his is he i s epo using
syn he ic pep ides o s udy NoV-speci ic T cell esponses in human subjec s and iden i y
T cell epi opes.
Keywo ds: no o i us, T cell epi ope, PBMC, VLP, pep ide pools, cellula immuni y, ELISPOT IFN-gamma
INTRODUCTION
No o i us (NoV) is ecognized as a leading cause o acu e gas oen e i is in child en and adul s
wo ldwide (Hall e al., 2016). Acco ding o a ecen ly published WHO epo , NoVs caused
120 million cases o dia heal diseases, mo e han any o he oodbo ne o in ec ious agen
(Ha elaa e al., 2015). These non-en eloped RNA i uses belonging o Calici i idae amily a e
highly in ec ious and cause equen ou b eaks ha can be se ious o indi iduals wi h unde lying
condi ions, he elde ly, and young child en (Hall e al., 2016). Cu en ly, he e is no cu e o
p e en i e accine a ailable agains NoV gas oen e i is.
Abb e ia ions: APC, allophycocyanin; BV, baculo i us; CEF, cy omegalo i us, Eps ein–Ba Vi us and In luenza i us;
CTL, cy o oxic T lymphocy e; ELISA, enzyme-linked immunoso ben assays; ELISPOT, enzyme-linked immunoso ben
spo ; FITC, luo escein iso hiocyana e; HBGA, his o-blood g oup an igen; HIV-1, human immunode iciency i us-1; HLA,
human leukocy e an igen; HRP, ho se adish pe oxidase; ICS, in acellula cy okine s aining; IFN-γ, in e e on gamma; IL-2,
in e leukin-2; NoV, No o i us; OD, op ical densi y; ORF, open eading ame; OVA, o albumin; PBMCs, pe iphe al blood
mononuclea cells; PBS, phospha e bu e ed saline; PE, phycoe y h in; SEB, s aphylococcal en e o oxin B; SFC, spo - o ming
cells; SYD, Sydney; Th, T helpe ; TNF-α, umo nec osis ac o alpha; VLP, i us-like pa icle.
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The ∼7.6 kb-long single-s anded posi i e-sense RNA genome
has h ee ORFs ha encode o he eplicase polyp o ein (ORF-
1), majo VP1 capsid p o ein (ORF-2), and mino capsid
p o ein VP2 (ORF-3; P asad e al., 1999). NoVs a e gene ically
classi ied in o se en genog oups (GI o GVII), which a e
u he di ided in o geno ypes based on capsid VP1 amino acid
sequence di e si y (K oneman e al., 2013;Vinje, 2015). NoV
geno ypes associa ed wi h human in ec ions belong p ima ily
o GI (9 geno ypes) and GII (22 geno ypes), wi h a >50%
di e gence o he VP1 a amino acid-le el (Zheng e al., 2006;
K oneman e al., 2013). Va ian s o he mos e icien ly e ol ing
GII.4 geno ype, wi h app oxima ely >95% homology in VP1
sequences, ha e p edomina ed o e he las wo decades (Bok
e al., 2009). VP1 consis s o a shell (S) domain, a hinge egion,
and a p o uding (P) domain, he la e o which is u he
di ided in o he P1 and ex emely a iable P2 subdomains;
hey con ain si es impo an o hos cell in e ac ion (Cao
e al., 2007). Nine y dime ic VP1 p o eins o m he ou e
laye o he icosahed al i us pa icle, which can a y in
size om ∼27 o 40 nm, depending on he geno ype (Vinje,
2015). VLPs a e sel -assembled by he ecombinan capsid
p o ein VP1. As a high-yield NoV cell cul u e sys em is
s ill elusi e (Jones e al., 2015), NoV VLPs a e essen ial o
s uc u al and immunogenici y s udies, and hey a e conside ed
p omising accine candida es (Blaze ic e al., 2011;A ma e al.,
2015).
No o i us in ec ions occu ea ly in li e (Hinkula e al.,
1995;Nu minen e al., 2011) and an ibody se op e alence
eaches almos 100% by adul hood (Jing e al., 2000;Ca mona-
Vicen e e al., 2015). Mul iple sequen ial in ec ions by gene ically
dis inc NoV s ains ha e been epo ed o occu equen ly,
especially in young child en (Sai o e al., 2014;Blaze ic e al.,
2015b). Acco ding o mos s udies, he du a ion o p o ec ion
in adul s is ela i ely sho and limi ed o gene ically simila
i us s ains (Wya e al., 1974;Johnson e al., 1990;Simmons
e al., 2013). The e is con o e sy o e he co ela es o
p o ec i e NoV immuni y and he leng h and speci ici y o he
p o ec ion, which is complica ed by he gene ic di e si y o
NoVs (Siebenga e al., 2009;Vinje, 2015) and di e ences in
he p e-exis ing NoV immuni y o humans (Lindesmi h e al.,
2010, 2015). Fu he mo e, he dis inc i e exp ession pa e n o
polymo phic HBGAs a ec s indi idual suscep ibili y o NoV
in ec ions (Lindesmi h e al., 2013). HBGAs, ound, e.g., on he
espi a o y and gas oin es inal ac su ace epi helia and in
bodily sec e ions, a e ecognized and bound by NoV pa icles
in a geno ype-speci ic manne (Uusi-Ke ula e al., 2014) and
pu a i ely se e as he ini ia ion si e o NoV in ec ion (Hu son
e al., 2002;Huang e al., 2003;Cao e al., 2007). Se um IgG
an ibody i e s blocking NoV VLP binding o HBGA ha e been
mos equen ly associa ed wi h p o ec ion om NoV in ec ion
and disease (Reeck e al., 2010;Nu minen e al., 2011;Malm
e al., 2014;A ma e al., 2015). Humo al immuni y o homo ypic
s ains is e icien ly elici ed (Malm e al., 2014), and b oadly
c oss- eac i e NoV-speci ic IgG an ibodies a e ound a e NoV
exposu e (Rockx e al., 2005;Lindesmi h e al., 2010;Malm e al.,
2014). Howe e , he induc ion o c oss-p o ec i e an ibodies
capable o blocking NoV VLP-HBGA binding in e ac ion ha e
been obse ed only o a ce ain deg ee be ween he di e en
geno ypes o he GII o GI genog oups, bu no ac oss he
genog oups (Reeck e al., 2010;A ma e al., 2015;Blaze ic
e al., 2015c). Addi ionally, mucosal IgA esponses ha e been
associa ed wi h he blocking ac i i y o NoV-HBGA binding
(Tamminen e al., 2016) and p o ec ion om in ec ion and
NoV gas oen e i is (Lindesmi h e al., 2003;Ramani e al.,
2015).
While mos NoV immuni y s udies ha e ocused on humo al
immune esponses (Ramani e al., 2016), only h ee s udies ha e
ga he ed in o ma ion on he ole o cell-media ed immuni y
(CMI), especially T cells in NoV in ec ion (Tacke e al., 2003;
Lindesmi h e al., 2005, 2010). Con ol o he pa hogenesis
wi h se e al i al in ec ions, such as HIV-1 (Rosenbe g e al.,
1997;Blaze ic e al., 2000;Sede e al., 2008), he in luenza
i us (Wilkinson e al., 2012;La G u a and Tu ne , 2014),
and he human papilloma i us (S anley, 2006), is known o
be dependen on T cell-media ed immuni y, especially CTLs.
The e o e, T cells a e also likely o play an impo an ole
in NoV clea ance. Following NoV in ec ion/challenge, IFN-γ
sec e ion by CD4+T cells has been analyzed only by ELISAs
using NoV VLPs as an igens, and he esul s indica ed he
gene a ion o NoV-speci ic memo y T cell esponses (Lindesmi h
e al., 2005, 2010). Fu he mo e, he essen ial ole o T
cells in con olling NoV in ec ions has been p o ided by
s udies wi h mu ine NoV (Chachu e al., 2008;Tomo e al.,
2013).
In he p esen s udy, we employed ELISPOT IFN-γand ICS
assays o s udy ex i o NoV-speci ic T cell esponses in adul
human subjec s o he i s ime. We used 18-me syn he ic
pep ides de i ed om he NoV GII.4 sequence o ganized in o
ma ix pep ide pools o iden i y NoV-speci ic T cell epi opes.
MATERIALS AND METHODS
Human Blood Dono s
Venous blood samples (30 ml) we e ob ained om eigh
heal hy adul (age ange 26–56, Table 1) olun ee s (labo a o y
pe sonnel) wi h no symp oms o acu e gas oen e i is in he
pas yea . Addi ional blood o wo olun ee s (Dono s 2 and 4)
was collec ed o subsequen ICS expe imen s. In o med consen
was ob ained om each olun ee p io o he sample collec ion
in acco dance wi h he Decla a ion o Helsinki. No app o al
by an e hical commi ee was needed. PBMCs we e isola ed
om he hepa inized blood samples wi hin 6 h o collec ion
by Ficoll-Paque PLUS (GE Heal hca e, Li le Chal on , UK)
densi y g adien and suspended in cell medium (CM) con aining
RPMI 1640 wi h Glu amaxR
and HEPES (GibcoTM by The mo
Fishe Scien i ic, Wal ham, USA) supplemen ed wi h 10 µg/ml
Gen amicin (GibcoTM) and 10% e al bo ine se um (FBS, Sigma–
Ald ich, S . Louis, MO, USA). Plasma was collec ed om each
dono ’s blood sample and s o ed a −20◦C un il u he use.
Human leukocy e an igen (HLA) yping was done by HLA locus-
speci ic ampli ica ion by PCR and he subsequen p obing o he
p oduc by sequence-speci ic oligonucleo ide p obe (PCR-SSOP,
P oImmune L d., Ox o d, UK).
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Malm e al. No o i us-Speci ic T Cell Responses
TABLE 1 | T cell in e e on gamma (IFN-γ) esponses o NoV an igens.
Dono # Age(yea s) CM GII.4-99 VLP GII.4 SYD VLP GI.3 VLP GII.4-99 pep ide poolaCEF pep ide pool OVA pep ide
1 38 13b80 63 128 90 355 15
2 35 0 10 15 8 268 225 0
3 56 3 50 53 90 80 1345 3
4 36 0 23 50 75 640 1390 3
5 47 0 18 23 20 28 763 3
6 44 3 60 33 38 95 1698 15
7 26 8 55 50 170 140 1248 8
8 45 10 50 35 40 58 325 18
aGII.4-99 comple e pep ide pool con aining 76 single pep ides. bMean spo o ming cell (SFC) alues pe 106PBMCs o pa allel wells in ELISPOT IFN-γassay. Posi i e
SFC (≥50 SFC) a e bolded. VLP, i us-like pa icle; CM, cell media; CEF, cy omegalo i us, Eps ein–Ba Vi us and In luenza i us; OVA, o albumin; SFC, spo - o ming
cells; SEM, s anda d e o o he mean.
Recombinan P o eins
No o i us VP1 genes o he ances o GII.4-1999 (GII.4-99,
e e ence s ain accession no: AF080551), he mos ecen GII.4
Sydney (SYD, accession no: AFV08795.1), and he ep esen a i e
geno ype o GI genog oup, GI.3-2002 (accession no: AF414403),
we e cloned in o pFas Bac1 ec o (In i ogen, Ca lsbad, CA,
USA) and ans ec ed in Spodop e a ugipe da (S )9 insec cells
(In i ogen) as desc ibed in de ail elsewhe e (Blaze ic e al.,
2011). The ampli ied BV s ocks we e used (mul iple o in ec ion,
MOI =1) o he p oduc ion o NoV VP1 p o eins in S 9 cells
which sel -assembled in o VLPs (Figu e 1). The ha es ed VLPs
we e pu i ied by wo consecu i e discon inuous suc ose g adien
ul acen i uga ions as p e iously desc ibed (Huh i e al., 2010).
The esidual suc ose was emo ed om he VLP p epa a ions
by dialysis agains a PBS ollowed by a concen a ion s ep wi h
Amicon Ul a-30 il e ing uni s (Me c Millipo e, Bille ica, MA,
USA). The p o ein concen a ions o he pu i ied VLP s ocks
we e de e mined by BCA P o ein Assay (Pie ceTM by The mo
Fishe Scien i ic). The pu i y, mo phology, and in i o and
in i o an igenici y o he VLPs we e analyzed by sodium dodecyl
sul a e polyac ylamide gel elec opho esis, elec on mic oscopy
(EM, Figu e 1), ELISA, and mouse immuniza ion expe imen s as
ea lie published by ou labo a o y (Huh i e al., 2010;Blaze ic
e al., 2011;Uusi-Ke ula e al., 2014;Malm e al., 2015).
Syn he ic Pep ides
Se en y-six indi idual syn he ic pep ides designa ed 99-1 o 99-
76 ep esen ing he en i e 539 amino acid (aa) sequence o GII.4-
99 NoV VP1 we e syn he ized (Synpep ide Co. L d, Shanghai,
China) as 18-me s wi h adjacen pep ides o e lapping by 11
aa. The pu i y exceeded 70%, as de e mined by high p essu e
liquid ch oma og aphy. Each lyophilized pep ide was dissol ed in
dime hyl sul oxide (DMSO, Sigma–Ald ich) and u he dilu ed
in s e ile PBS (Lonza, Basel, Swi ze land) o use in he assays.
The amoun o DMSO ne e exceeded 0.01% in he inal
medium. A comple e pep ide pool (con aining all 76 pep ides)
and 18 wo-dimensional ma ix pools (M1–M18) we e made
om indi idual pep ides as p e iously desc ibed (Wilkinson
e al., 2012). The i s dimension included nine pools (M1–M9),
he second dimension also included nine pools (M10–M18), and
each pool con ained eigh o nine indi idual pep ides so ha each
pep ide was p esen in wo di e en ma ix pools. A CEF pep ide
pool (CTL Eu ope GmbH) con aining 32 i al T cell epi opes
om he human cy omegalo i us i us, he Eps ein–Ba i us,
and he in luenza i us was used as a posi i e con ol pep ide
pool (Cu ie e al., 2002) in he ELISPOT assays. A mu ine T
cell epi ope (McFa land e al., 1999) de i ed om OVA (323–
339, In i ogen, San Diego, CA, USA) was used as an i ele an
nega i e con ol pep ide in he assays.
No o i us-Speci ic Se ology Assay
An ELISA was u ilized o de ec he NoV GII.4-99-, GII.4
SYD-, and GI.3-speci ic IgG an ibodies in he human samples
as p e iously desc ibed (Malm e al., 2014). In b ie , plasma
dilu ed se ially s a ing a 1:100 was added o NoV VLP-coa ed
(25 ng/well) hal -a ea 96-well pla es (Co ning Inc., Co ning, NY,
USA) and incuba ed o 1h a +37◦C. One known NoV-posi i e
and -nega i e human se um was added o each pla e as con ols.
Blank wells lacking a sample we e added as backg ound con ols.
The bound an ibodies we e de ec ed by HRP conjuga ed an i-
human IgG (No exR
by The mo Fishe Scien i ic) eac ing wi h
o-phenylenediamine dihyd ochlo ide subs a e (OPD, Sigma-
Ald ich). The OD alues o each well we e measu ed in a
mic opla e eade (Vic o 21420, Pe kin Elme , Wal ham, MA,
USA). The esul s a e exp essed as he end-poin i e s o he
indi idual plasma de e mined as he highes i e gi ing an
OD alue abo e he se cu -o alue (mean OD o nega i e
con ol wells +3×SD and a leas 0.100 OD) a e backg ound
sub ac ion.
ELISPOT IFN-γAssay
F esh PBMCs o he eigh dono s we e assayed in an ELISPOT
assay o IFN-γp oduc ion upon s imula ion wi h NoV VLPs
(GII.4-99, GII.4 SYD, and GI.3) o syn he ic GII.4-99 pep ides
used as a comple e pep ide pool, pep ide ma ices, and in
subsequen expe imen s as single pep ides. Nine y-six-well
ni ocellulose il e pla es (Millipo e) we e coa ed a +4◦C (18–
72 h) wi h an i-human IFN-γcap u e an ibody (Mab ech) a
a concen a ion o 5 µg/ml. A e washing wi h s e ile PBS,
he pla es we e blocked o 2–3 h wi h CM con aining 10%
FBS. The an igens we e added o he pla es: VLPs a inal
concen a ions o 0.5 and 5 µg/ml and GII.4-99 pep ide pools
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Malm e al. No o i us-Speci ic T Cell Responses
FIGURE 1 | Elec on mic oscopy image o baculo i us-insec cell sys em-p oduced and suc ose g adien -pu i ied no o i us GII.4-99 (A), GII.4 Sydney
(B), and GI.3 (C) i us-like pa icles (VLPs). VLPs we e nega i ely s ained wi h 3% u anyl ace a e (pH 4.5) and he p epa a ions we e examined using an FEI
Tecnai F12 elec on mic oscope a a magni ica ion o 23,000×.
a 2 µg/ml. The dilu ions o he inal pep ide pools used in he
assay we e de e mined om ea lie i a ion expe imen s. The
single pep ides we e used a 0.5–4 µg/ml inal concen a ion.
Posi i e con ol (CEF-pool, a 1.5 µg/ml) and nega i e con ol
(OVA 323–339, a 2 µg/ml) pep ides and CM only (backg ound
con ol) we e es ed on each pla e. F esh PBMCs we e added a
0.2 ×106cells/well and he pla es we e incuba ed o 44 h a
+37◦C and 5% CO2. A e disca ding he cells, he pla es we e
washed and bio inyla ed an i-human IFN-γan ibody (Mab ech)
was added a 2 µg/ml. The pla es we e incuba ed o 3 h a RT
ollowed by 1 h incuba ion wi h 1:500 dilu ed s ep a idin-HRP
(BD, T en on, NJ, USA). The spo s we e de eloped o 7.5 min
wi h Vec o No a Red subs a e (Vec o Labs, Bu lingame,
CA, USA) in he da k and he eac ion was s opped wi h
ap wa e . The pla es we e ai -d ied p io o au oma ed spo
coun ing by an ImmunoSpo Se ies II analyze (CTL Eu ope,
Lein elden-Ech e dingen, Ge many). The esul s a e exp essed as
mean SFC/106PBMCs o he duplica e wells. The esul s we e
conside ed posi i e i he numbe o SFC pe well was ≥50
spo s/106PBMCs and wice abo e he backg ound con ol (CM
only wells). SFC in he backg ound con ol wells ne e exceeded
15 SFC/106PBMCs.
In acellula Cy okine S aining (ICS)
An ICS assay was employed o measu e IFN-γ, TNF-γand IL-
2 cy okine p oduc ion, and o de e mine he T cell pheno ype
esponsible o cy okine sec e ion. F esh PBMCs o wo dono s
we e s imula ed (1 ×106cells/condi ion) wi h 2 µg/ml 99–20
single pep ide o wi h 1 µg/ml SEB (Sigma) in he p esence o
1µg/ml CD28 and 1 µg/ml CD49d cos imula o y an ibodies
(BD Biosciences, San Jose, CA, USA) o 2 h a 37◦C. As a
con ol, PBMCs we e incuba ed in CM only supplemen ed wi h
he cos imula o y an ibodies. The p o ein anspo inhibi o
b e eldin A (GolgiPlug, BD Biosciences, San Jose, CA, USA) a
a concen a ion o 10 µg/ml was added and he incuba ion was
con inued o 16 h. A e he s imula ion, cells we e ea ed wi h
EDTA o 15 min o a es ac i a ion and emo e adhe en cells,
and washed wi h FACS S ain bu e . Non-speci ic binding was
blocked by incuba ing he cells o 10 min wi h Human BD Fc
Block. Cell su ace ma ke s CD3 and CD8 we e s ained be o e
he pe meabiliza ion o he cells by incuba ing he cells wi h
monoclonal an ibodies agains human CD3 (clone UCHT1, FITC
conjuga e) and CD8 (clone RPA-T8, Pe CP-Cy5.5 conjuga e)
in 50 µl S ain bu e o 30 min on ice in he da k. Cells
we e washed wice wi h S ain bu e and ea ed wi h BD
Fixa ion/Pe meabiliza ion solu ion o 20 min acco ding o he
manu ac u e ’s ins uc ions. Fixed and pe meabilized cells we e
washed wice wi h BD Pe m/Wash bu e be o e in acellula
s aining wi h he mix u e o IFN-γ(clone 4S.B3) phycoe y h in
(PE)-Cy7-conjuga e, IL-2 (clone MQ1-17H12) phycoe y h in-
conjuga e, and TNF-α(clone MAb11) APC-conjuga e in 50 µl
Pe m/Wash bu e o 30 min on ice in he da k. A e he ICS
incuba ion pe iod, PBMCs we e washed wice wi h Pe m/Wash
bu e and esuspended in S aining Bu e o acquisi ion and
analysis. All eagen s used o ICS we e pu chased om BD
Pha mingen (San Jose, CA, USA).
Flow Cy ome ic Analysis
Samples we e acqui ed using a 2-lase FACS Can oII low
cy ome e (BD) and FACSDi a So wa e V 6.1.3 (Bec on
Dickinson, Heidelbe g, Ge many) wi hin 2 h o s aining wi h
luo escen an ibodies. PMT ol ages we e adjus ed wi h he
uns ained, ixed PBMC sample. A leas 70,000 CD3+cells
we e acqui ed pe sample. The ga es we e designed in he
nega i e con ol sample wi h conside a ion o he ma ke
down egula ion om he s imula ed samples. The ga ing
s a egy is shown in Figu e 2. The inal analysis plo s o
IFN-γIL-2, and TNF-αwe e ga ed on CD3+CD8+and
CD3+CD8−popula ions. The da a analysis was pe o med
using FlowJo so wa e e sion 10.1 (T ee S a , San Ca los,
CA, USA). Cy okine esponses we e de ined as posi i e i
he pe cen ages o cy okine-posi i e cells s imula ed wi h he
an igens we e a leas wo imes highe and >0.05% abo e he
cy okine posi i e cells in he backg ound (CM; Fe a i e al.,
2004).
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FIGURE 2 | The ga ing s a egy o low cy ome y analysis om ep esen a i e da a. PBMCs om a dono we e s imula ed wi h SEB o 16 h in he
p esence o b e eldin A and hen s ained o IFN-γand lineage ma ke s CD3 and CD8. A o wa d and side sca e was used o ga ing he lymphocy e popula ion.
CD3+lymphocy e popula ion was u he ga ed o CD8+and CD8−popula ions and IFN-γs aining was analyzed o bo h popula ions as p e iously desc ibed by
o he s (Meddows-Taylo e al., 2007). Do plo s show ga ed popula ions and e en s as pe cen ages o he pa en popula ion.
S a is ical Analyses
Fishe ’s exac es was used o compa e he di e ences in he
IgG end-poin i e s. The Mann–Whi ney U- es was used o
assess he di e ences be ween NoV VLP- and GII.4-99 pep ide
pool-induced SFC/106PBMCs in he ELISPOT. Spea man’s
ank co ela ion coe icien was used o examine he co ela ion
be ween he SFC/106PBMCs and he an ibody end-poin i e .
S a is ical analyses we e pe o med using IBM SPSS S a is ics
(SPSS, Chicago, IL, USA) e sion 23.0. S a is ical signi icance
was de ined as p<0.05. All hypo hesis es s we e wo-
ailed.
RESULTS
An ibody Responses
NoV GII.4-99, GII.4 SYD, and GI.3 VLPs we e used as an igens
in an ELISA o es plasma IgG an ibody binding o na i e,
con o ma ional an igenic de e minan s. The esul s showed ha
all eigh dono s had NoV-speci ic an ibodies (Figu e 3A),
indica ing p e ious NoV exposu e, bu he magni ude o he
geno ype-speci ic esponses a ied among he dono s. All dono s
had an ibody esponse o GII.4-99, and he end-poin i e s o his
geno ype we e he highes o he h ee VLPs es ed (Figu e 3B,
p<0.05).
T Cell Response o NoV VLPs and Capsid
Pep ide Pool
An ELISPOT assay was u ilized o de ec ex i o IFN-γ elease
om human PBMCs a e in i o s imula ion o 44 h wi h
NoV VLPs (GII.4-99, GII.4 SYD, and GI.3) o a comple e pep ide
pool de i ed om GII.4-99 capsid VP1. None o he dono s
esponded o s imula ion wi h 0.5 µg/ml VLPs (da a no shown).
Table 1 shows ha when using a high concen a ion (5 µg/ml)
o he VLPs, six o he eigh dono s esponded o a leas one
VLP, and h ee dono s p oduced IFN-γin esponse o all VLPs
es ed. In e es ingly, all bu one dono (#5) esponded o he
GII.4-99 pep ide pool, bu only i e esponded o GII.4-99 VLPs
(Table 1). Fu he mo e, he numbe o IFN-γ-sec e ing cells
in esponse o he pep ide pool was highe (mean 175 ±71.2
SFC/106cells) han he esponse o he co esponding GII.4-
99 VLPs (mean 43 ±8.5 SFC/106cells; p<0.05). Howe e ,
he e was a di e ence in he magni ude o hese esponses
in each indi idual and high a ia ion among he dono s was
seen, especially wi h he pep ide pool s imula ion (58–640 mean
SFC/106PBMCs). Two dono s (#2 and #4), who we e he
bes esponde s o he GII.4-99 pep ide pool, had no esponse
o he VLPs o he same o igin. Only one dono (#5) did
no espond ei he o he VLP o pep ide pool s imula ion.
PBMCs o all eigh dono s p oduced IFN-γa e he CEF
pep ide pool s imula ion (>200 SFC/106PBMCs) and none
esponded o he nega i e OVA con ol pep ide (<20 SFC/106
PBMCs).
Co ela ion o T Cell and An ibody
Responses
Nex , we de e mined whe he NoV VLP-speci ic an ibody i e s
co ela ed wi h he co esponding geno ype T cell esponses
de ec ed in he ELISPOT IFN-γassays (Figu e 4). No co ela ion
(p>0.05) was obse ed be ween GII.4-99 an ibody i e s and
IFN-γ-p oducing PBMC numbe s a e he VLP (Figu e 4A)
o comple e pep ide pool (Figu e 4B) s imula ion. Simila ly,
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FIGURE 3 | No o i us geno ype-speci ic immunoglobulin G (IgG) an ibody i e s. The plasma o eigh dono s was used o de e mine indi idual end-poin i e
(A) and geome ic mean i e s (GMTs) wi h 95% con idence in e als (B) agains NoV GII.4-1999, GII.4 Sydney, and GI.3 VLPs in an enzyme-linked immunoso ben
assay (ELISA). S a is ical signi icance was de e mined by Fishe ’s exac es , and a p- alue ≤0.05 was conside ed o be s a is ically signi ican (∗).
FIGURE 4 | Co ela ion o GII.4-1999-speci ic an ibody and T cell esponses. Spea man’s ank co ela ion ( ) was de e mined be ween he no o i us
GII.4-1999-speci ic an ibody i e and ELISPOT IFN-γspo - o ming cell (SFC)/106pe iphe al blood mononuclea cells (PMBCs) in esponse o GII.4-99 VLPs (A) and
a comple e capsid pep ide pool (B) s imula ion.
no co ela ion was de ec ed be ween an ibody i e s and IFN-
γ-sec e ing cell numbe s agains GII.4 SYD ( = −0.268,
p=0.521) o GI.3 ( =0.328, p=0.618) VLPs (da a no shown).
Mapping o T Cell Epi opes wi h Ma ix
Pep ide Pools
Se en y-six 18-me o e lapping pep ides spanning he en i e
GII.4-99 VP1 we e used o iden i y NoV-speci ic T cell epi opes.
PBMCs o each dono we e assayed o IFN-γp oduc ion in an
ELISPOT assay in esponse o he pep ides o ganized in o 18
ma ix pools (M1–M18). Only h ee o he eigh dono s (dono s
#2, #4, and #7) esponded o a leas wo ma ix pools (Figu e 5),
and he same dono s also had he s onges esponse o he
comple e GII.4-99 pep ide pool (Table 1). The s onges pep ide-
speci ic IFN-γ esponse was owa d ma ix pools M2 and M12 in
dono s #2 and #4. Dono #4 also esponded mode a ely o ma ix
pools M7 and M11, as did dono #7 o M4 and M13 (Figu e 5).
Fo each dono esponding o he ma ix pools, s imula o y
pep ides con ained in he pools we e decon olu ed om a wo-
dimensional ma ix sys em, whe e each pep ide appea ed only
once in each dimension. Th ee single pep ides we e iden i ied as
pu a i e T cell epi opes: 99-16 pep ide (aa 106-123) included in
he M7 and M11 pools (dono #4), 99-20 pep ide (aa 134-151)
included in he M2 and M12 pools (dono s #2 and #4), and 99-31
(aa 211-228) in he M4 and M13 pools (dono #7).
T Cell Res ic ion o he Epi opes
In he h ee dono s wi h he T cell epi opes decon olu ed
om a wo-dimensional ma ix sys em, newly isola ed PBMCs
we e s imula ed wi h he single pep ides 99-16, 99-20, and
99-31 in he second ELISPOT IFN-γassay. Only pep ide
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FIGURE 5 | Enzyme-linked immunoso ben spo in e e on gamma (IFN-γ) esponses o GII.4-1999 ma ix pep ide pool s imula ion. Pe iphe al blood
mononuclea cells (PBMCs) o eigh dono s (#1-8) we e s imula ed wi h 76 18-me GII.4-99 capsid-de i ed single pep ides o ganized in o 18 ma ix pools (M1–M18)
o le uns imula ed (CM, cul u e media). Shown a e he mean spo - o ming cells (SFC)/106PBMCs o wo eplica e wells wi h he s anda d e o s o he mean. The
cu -o line (do ed line) ep esen s he posi i e SFC/106 alue o >50 SFC/106cells.
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FIGURE 6 | Enzyme-linked immunoso ben spo IFN-γ esponses owa d NoV GII.4-1999 T cell epi ope 99-20. F esh pe iphe al blood mononuclea cells
(PBMCs) o dono s #2 (A) and #4 (B) we e s imula ed wi h an inc easing concen a ion (0–4 µg/ml) o pep ide 99-20 con aining he pu a i e NoV-speci ic T cell
epi ope. Shown a e he spo - o ming cells (SFC)/106PBMCs o wo eplica e wells wi h s anda d e o s o he mean. The cu -o line (do ed line) ep esen s he
posi i e SFC/106 alue o >50 SFC/106cells.
99-20 (134SPSQVTMFPHIIVDVRQL151) induced s ong IFN-γ
p oduc ion in dono s #2 (Figu e 6A) and #4 (Figu e 6B), who
o iginally esponded s ongly o he M2 and M12 ma ix pools
(Figu e 5 and da a no shown).
Pep ide 99-20 was u he es ed by ICS o iden i y he T cell
subse esponsible o IFN-γp oduc ion in dono s #2 and #4
de e mined by he ELISPOT assay. Addi ionally, IL-2 and TNF-α
cy okine sec e ion we e simul aneously analyzed. F esh PBMCs
we e s imula ed wi h he pep ide 99-20 o SEB as a posi i e
con ol an igen. The uns imula ed PBMCs (CM) sample se ed
as a backg ound con ol. The pe cen ages o cy okine-p oducing
cells we e de e mined wi hin he CD3+CD8+and CD3+CD8−
(CD4+) T cell popula ions. Pep ide 99-20 induced he exp ession
o all h ee cy okines by CD3+CD8+T cells o bo h dono s
(Figu e 7), iden i ying he epi ope as CD8+T cell- es ic ed.
These wo dono s we e u he yped o he HLA-A, -B, and
-C alleles in o de o iden i y possible common HLA class I
es ic ing molecule binding o he pep ide epi ope 99-20. The
yping esul s showed ha bo h dono s ca y HLA-A∗02:01 allele.
Fu he analysis o he cells esponding o pep ide 99-20 by
IFN-γp oduc ion (ga ing on CD3+CD8+IFN-γ+lymphocy es)
e ealed ha ∼74% o hese popula ions in bo h dono s we e also
simul aneously exp essing IL-2 and TNF-αcy okines. No posi i e
esponses we e ound in he CD4+T cell ga e o any o he
cy okines a e he 99-20 pep ide s imula ion (da a no shown).
Bo h he CD3+CD8+and CD3+CD8−T cell popula ions o
each dono we e posi i e o all cy okines a e SEB s imula ion,
con i ming he good iabili y and unc ionali y o he cells and a
success ul s aining p o ocol (Figu e 2 and da a no shown).
DISCUSSION
Ve y limi ed published in o ma ion is a ailable on na u al T
cell immuni y in NoV-in ec ed humans (Tacke e al., 2003;
Lindesmi h e al., 2005, 2010). CD4+Th and CD8+CTLs
a e impo an in p o ec ion om in ec ion by di e en i uses,
including in luenza (La G u a and Tu ne , 2014;La ey e al.,
2015), HIV (Rosenbe g e al., 1997;Blaze ic e al., 2000), HPV
(S anley, 2006), and he Eps ein–Ba i us (Peppe l e al.,
1998). P o ec i e immuni y o NoV migh also be pa ially
dependen on he ac i a ion o T cell immuni y. Mu ine NoV
es ablishes pe sis en in ec ion in he absence o unc ional T
cells, suppo ing he impo ance o T cell-media ed immuni y in
NoV in ec ion (Wobus e al., 2006;Chachu e al., 2008;Tomo
e al., 2013).
We s udied an ibody and T cell-media ed p e-exis ing
immune esponses in eigh heal hy adul dono s. Each dono
had a posi i e se um IgG an ibody i e agains all NoV
VLPs es ed, indica ing p e ious exposu es o he NoV (Jing
e al., 2000;Nu minen e al., 2011;Ca mona-Vicen e e al.,
2015). Two NoV GII.4 VLPs, de i ed om an ances o GII.4-
99 and he mos ecen GII.4 SYD a ian s, we e chosen as
an igens, as GII.4 has been he domina ing NoV geno ype o
o e wo decades (Bull and Whi e, 2011;Eden e al., 2014).
GI.3 VLPs we e chosen as a ep esen a i e geno ype o he
GI genog oup con ibu ing o a mino i y o NoV in ec ions
in humans (Ma hews e al., 2012). The s onges an ibody
esponse in se en o he eigh dono s was de ec ed owa d
GII.4-99 VLPs, sugges ing ha mos o he dono s ha e been
exposed o GII.4-99 o an igenically closely ela ed a ian s in
he pas . As hese dono s ha e likely no expe ienced in ec ion
wi h he ecen ly ci cula ing GII.4 SYD, he an ibody esponses
o his geno ype we e signi ican ly lowe , p obably consis ing
o c oss- eac i e an ibodies aised o he ea lie ci cula ing
a ian s o he GII.4 geno ype (Rockx e al., 2005;Malm e al.,
2015).
Di e en me hods a e employed o measu e T cell esponses
in na u al in ec ions and accina ion s udies. ELISPOT and ICS
de ec ing IFN-γsec e ion om PBMCs induced by syn he ic
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FIGURE 7 | Exp ession o IFN-γ, IL-2, and TNF-αby in acellula s aining a e s imula ion wi h NoV GII.4-1999 pep ide epi ope 99-20. Pe iphe al blood
mononuclea cells (PBMCs) o dono s #2 ha ing A∗01:01/∗02:01; B∗08:01/∗56:01; C∗01:02/∗07:01 HLA class I pheno ype alleles (A) and #4 ha ing
A∗02:01/∗32:01; B∗35:01/∗40:02; C∗03:04/∗04:01 HLA class I pheno ype alleles (B) we e s imula ed wi h 2 µg/ml o 99-20 single pep ide o 16 h and he cy okine
esponse was measu ed by low cy ome y. PBMCs in cul u e media only (CM) (le panel) and pep ide-s imula ed PBMCs ( igh panel) we e ga ed o CD3+CD8+T
cells and he pe cen ages o IFN-γ, IL-2, o TNF-αcy okine-exp essing cells a e indica ed.
pep ides and/o ecombinan whole p o eins a e he mos
commonly used (Schmi el e al., 2001;Cu ie e al., 2002;
Ku sche e al., 2008;S eeck e al., 2009;F ee and Rindi, 2013;
Ku sche e al., 2013;La ey e al., 2015). In compa ison o
ICS, ELISPOT is highly sensi i e bu does no iden i y he cell
ype esponsible o he cy okine p oduc ion (Tassignon e al.,
2005;Tobe y e al., 2006). The e o e, using ICS o complemen
esul s om an ELISPOT assay is a wo hwhile and gene ally
used p ac ice (F ee and Rindi, 2013;La ey e al., 2015).
To s udy T cell-media ed immuni y, we in es iga ed ex i o
IFN-γp oduc ion om eshly isola ed PBMCs in esponse o
s imula ion wi h NoV VLPs and GII.4-99 syn he ic pep ides by
bo h ELISPOT IFN-γand ICS. We de ec ed a much highe
equency and magni ude o T cell esponses using an ELISPOT
IFN-γassay in p e iously exposed adul human dono s owa d
syn he ic pep ides (pooled and indi idual) han he whole p o ein
assembled in he VLPs. All bu one dono esponded o he
comple e GII.4-99 pep ide pool, bu only i e esponded o GII.4-
99 VLPs. Fu he mo e, wo dono s had a s ong CD8+T cell
esponse o GII.4-99-speci ic pep ide 99-20, bu no esponse o
he co esponding VLPs, as shown by he ELISPOT IFN-γ.
The g ea e esponse o he pep ides han VLPs de ec ed
he e is no su p ising, as he 15-20-me syn he ic pep ides
added exogenously o he PBMCs ci cum en he need o
an igen p ocessing by di ec ly binding o HLA classes I and
II molecules and s imula ing bo h CD4+and CD8+T cells
e icien ly (Blaze ic e al., 1995;Ke n e al., 2000;Kau hold
e al., 2005). In con as , p o ein an igens equi e p ocessing
in o smalle pep ide agmen s by an igen-p esen ing cells be o e
being p esen ed o T cell ecogni ion, p e e en ially s imula ing
CD4+T cells h ough HLA class II molecules. The ELISPOT
assay employing pep ides usually equi es o e nigh incuba ion
(Schmi el e al., 2001;Kau hold e al., 2005). The e o e, we used
a 44 h assay o ensu e p ocessing o he VLPs and he binding
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