ORIGINAL RESEARCH
published: 27 No embe 2018
doi: 10.3389/ immu.2018.02782
F on ie s in Immunology | www. on ie sin.o g 1No embe 2018 | Volume 9 | A icle 2782
Edi ed by:
Linda F. Van Dyk,
Uni e si y o Colo ado Den e ,
Uni ed S a es
Re iewed by:
Lijun Xin,
Cincinna i Child en’s Hospi al Medical
Cen e , Uni ed S a es
Jo ma Hinkula,
Linköping Uni e si y, Sweden
*Co espondence:
Vesna Blaze ic
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Vi al Immunology,
a sec ion o he jou nal
F on ie s in Immunology
Recei ed: 05 July 2018
Accep ed: 12 No embe 2018
Published: 27 No embe 2018
Ci a ion:
Malm M, Vesika i T and Blaze ic V
(2018) Iden i ica ion o a Fi s Human
No o i us CD8+T Cell Epi ope
Res ic ed o HLA-A∗0201 Allele.
F on . Immunol. 9:2782.
doi: 10.3389/ immu.2018.02782
Iden i ica ion o a Fi s Human
No o i us CD8+T Cell Epi ope
Res ic ed o HLA-A∗0201 Allele
Ma ia Malm, Timo Vesika i and Vesna Blaze ic*
Facul y o Medicine and Li e Sciences, Vaccine Resea ch Cen e , Uni e si y o Tampe e, Tampe e, Finland
No o i us (NoV) causes a subs an ial global bu den o acu e gas oen e i is in all
age g oups and he de elopmen o NoV accine is a high p io i y. The e a e s ill
gaps in unde s anding o p o ec i e NoV-speci ic immuni y. An ibody media ed immune
esponses ha e been widely s udied, bu in con as , he esea ch on NoV-speci ic
human T cell-media ed immuni y is e y limi ed. We ha e ecen ly epo ed NoV capsid
VP1-speci ic 18-me pep ide (134SPSQVTMFPHIIVDVRQL151) o induce s ong CD8+
T cell immune esponses in heal hy adul dono s. This wo k ex ends o iden i y he
p ecise NoV T cell epi ope and he es ic ing human leucocy e an igen (HLA). Pen ame
echnology was used o de ec HLA-A∗0201- es ic ed T cell-media ed esponses o
10-me pep ide 139TMFPHIIVDV148 o ou heal hy adul blood dono s. Immunogenici y
o he 10-me epi ope was con i med by ELISPOT IFN-γand in acellula cy okine
s aining (ICS) on low cy ome y. A popula ion o CD3+CD8+T lymphocy es binding
o HLA-A∗0201/TMFPHIIVDV pen ame s was iden i ied in wo HLA-A∗0201-posi i e
dono s. Recogni ion o he 10-me epi ope by T cells esul ed in a s ong IFN-γsec e ion
as shown by ELISPOT assay. In addi ion, ICS con i med ha high p opo ion (31 and
59%) o he TMFPHIIVDV epi ope- esponsi e CD3+CD8+T cells in he wo dono s had
mul i unc ional pheno ype, simul aneously p oducing IFN-γ, IL-2 and TNF-αcy okines.
In he p esen s udy no el human NoV HLA-A∗0201- es ic ed minimal 10-me epi ope
139TMFPHIIVDV148 in he capsid VP1 was iden i ied. The HLA-pep ide pen ame s aining
o T cells om heal hy dono PBMCs and cy okine esponses in ex- i o ELISPOT and
ICS assays sugges ha his epi ope is ecognized du ing NoV in ec ion and ac i a es
memo y pheno ype o he epi ope-speci ic mul i unc ional CD8+T cells. The impo ance
o his epi ope in p o ec ion om NoV in ec ion emains o be de e mined.
Keywo ds: no o i us, CD8 T cell epi ope, HLA-A2∗0201, mul i unc ional T cells, pen ame , ELISPOT IFN-gamma,
cellula immuni y
INTRODUCTION
No o i uses (NoV) a e g oup o e y con agious i uses ha cause >90% o non-bac e ial
and app oxima ely hal o all-cause epidemic gas oen e i is wo ldwide. NoV gas oen e i is is a
signi ican public heal h p oblem wi h high clinical and economic cos s, ha is es ima ed o cause
o e 200,000 dea hs annually, mainly in esou ce-limi ed coun ies (1). Despi e scien i ic e o s
and inc easing awa eness o NoV bu den, he e is no accine a ailable ye (2,3). NoV exhibi s a
g ea gene ic di e si y wi h six cu en ly ecognized genog oups (GI-GVI), human NoVs belonging
Malm e al. Human No o i us T Cell Epi opes
mainly o GI and GII, ha include o e 30 geno ypes and
nume ous in a-geno ype a ian s (4). GII.4 geno ype has been
p edominan since he mid-90s, including pandemic a ian s
US 1995/96, Fa ming on Hills 2002, Hun e 2004, New O leans
(NO) 2009, and mos ecen ly, Sydney 2012 (5). GII.4 a ian s
sha e >95% o hei VP1 capsid amino acid (aa) sequence,
whe eas <85% o he capsid aa a e iden ical be ween di e en
geno ypes (6). The an igenic di e si y o NoVs is likely o
ha e majo impac on e ading hos immune esponses and
complica es designing o NoV accines. NoV esea ch is u he
limi ed due o he lack o small animal model and e icien NoV
p opaga ion in cell cul u e (7,8), bu spon aneously o med
NoV VP1 i us-like pa icles (VLPs) a e success ully u ilized as
i al su oga es o assaying immune esponses and as accine
candida es (9,10).
NoV in ec ions cause acu e bu sel -limi ed illness al eady
a ea ly age and se e al consecu i e in ec ions occu equen ly
(11–13). P o ec i e immuni y and clea ance o NoV in ec ions
a e no well-cha ac e ized (1,2). E en hough he e is some
indica ion o p e-exis ing an ibodies con e ing p o ec ion (14)
he le el o se um NoV-speci ic an ibodies is no di ec ly
linked o p o ec ion om in ec ion (15). The bes co ela e o
p o ec ion om NoV in ec ion iden i ied so a a e blocking
(neu alizing) an ibodies, ha a e able o block he binding o
NoV VLPs o he pu a i e ecep o s, his o-blood g oup an igens
(HBGA’s) (15). NoV-speci ic an ibodies a e c oss- eac i e o
some ex en wi hin genog oups howe e , blocking an ibodies
a e mos ly geno ype-speci ic wi h low p o ec i e capaci y agains
mo e dis inc s ains (1). Al hough NoV-speci ic an ibodies a e
ex ensi ely s udied e y li le is published on human T cell
immuni y o NoV and i s’ ole in p o ec ion (16–19). S udies
using a mu ine no o i us (MNV)-mouse model ha e sugges ed
T cell-media ed immuni y o be impo an in clea ance o
MNV in ec ion (20,21). To his end, cy o oxic CD8+T cell-
media ed immune esponses may p o ide subs an ial p o ec ion
agains se ologically dis inc i uses ia ecogni ion o c oss-
eac i e, conse ed epi opes, such as obse ed in in luenza i us
in ec ion (22).
Using ma ix pep ide app oach we ha e ecen ly iden i ied a
NoV-speci ic 18-me pep ide (134SPSQVTMFPHIIVDVRQL151)
con aining CD8+T cell epi ope likely es ic ed o human
leukocy e an igen (HLA)-A∗0201 allele (17). Fu he mo e,
pep ide binding p edic ion ools ha e p edic ed 10 amino acid
(aa) s e ch as a minimal epi ope (139TMFPHIIVDV148). In he e,
expe imen s we e unde aken o expe imen ally con i m he
ecogni ion o he 10-me epi ope and i s p esen a ion by he
HLA-A∗0201 allele.
MATERIALS AND METHODS
Blood Dono s and Cell Isola ion
Fou heal hy adul s (age 35–45, labo a o y pe sonnel)
olun ee ing in a s udy ecen ly conduc ed by ou labo a o y
we e selec ed based on hei CD8+T cell esponses o NoV GII.4
VP1-speci ic 18-me pep ide 134SPSQVTMFPHIIVDVRQL151
(o iginally named 99-20) ecen ly desc ibed (17). The wo
pep ide esponde s we e HLA yped as HLA-A∗02:01
ca ie s (17). The cells o he wo dono s iden i ied as non-
esponde s o he 18-me pep ide we e used as nega i e
con ols. Pe iphe al blood mononuclea cells (PBMCs) o he
hepa in blood sample we e ob ained by Ficoll-Pague PLUS
(GE Heal hca e, Li le Chal on , Uni ed Kingdom) densi y
g adien cen i uga ion. PBMCs we e ozen in 10% DMSO
in e al bo ine se um (FBS) using eezing con aine (M .
F os yTM, The mo Scien i ic,Wal ham, MA, Uni ed S a es) wi h
con olled a e o cooling a −80◦C and ans e ed o liquid
ni ogen. P io o analysis, PBMCs we e hawed, washed, and
esuspended in cul u e medium (CM) con aining RPMI 1640
wi h Glu amax R
and HEPES (GibcoTM by The mo Fishe
Scien i ic) 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, Uni ed S a es). The samples es ed he e a e collec ed a
a single bleed. Each sample aliquo was es ed simul aneously
by pen ame s aining, enzyme-linked immunoso ben spo
in e e on-gamma (ELISPOT IFN-γ) assay and in acellula
cy okine s aining (ICS) assays, a leas wo imes. W i en
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 hics commi ee was equi ed as pe he local
legisla ion.
Syn he ic Pep ides and Pen ame s
To p edic he op imal HLA-A∗02:01 allele binding
epi ope wi hin he 18-me NoV-speci ic pep ide
134SPSQVTMFPHIIVDVRQL151, a i icial neu al ne wo ks
(ANN) (23) implemen ed a Immune Epi ope Da abase and
Analysis Resou ce (IEDB) was employed (24–26). A 10-me high
binding a ini y sequence 139TMFPHIIVDV148 was iden i ied
(17) and he pep ide was syn he ized wi h pu i y >75%
(Synpep ide Co. L d, Shanghai, China). SYFPEITHI p edic ion
algo i hm (h p://www.sy pei hi.de) (27) was used o sco ing
he pep ide a ini y o HLA-A∗0201 allele, as a binding sco e
o >21 ensu es syn hesis o a cus om pen ame by P oimmune
L d. (Ox o d, Uni ed Kingdom). HLA-A∗0201/TMFPHIIVDV
pen ame (P o5 R
MHC Class I Pen ame ) labeled wi h R-
phycoe y h in (R-PE) was syn he ized by P oimmune L d. In
addi ion, HLA-A∗0201 nega i e con ol pen ame conjuga ed
o R-PE was syn he ized as well. The deg ee o conse a ion
o he 10-me epi ope among di e en NoV geno ypes and
genog oups was in es iga ed using Basic Local Alignmen Sea ch
Tool (BLAST) o sequence iden i y. E olu iona y analyses
o he majo capsid VP1 aa sequence o he aligned NoV
geno ypes we e conduc ed in MEGA X (28). The e olu iona y
dis ances we e compu ed using he Poisson co ec ion
me hod (29).
Pen ame S aining
PBMCs we e ea ed o 10 min wi h Human BD Fc Block
o p e en non-speci ic s aining. To disc imina e iable om
non- iable cells, Ho izon Fixable Viabili y s ain 780 was
used acco ding o manu ac u e ’s ins uc ions. The cells (1
×106) we e incuba ed o 30 min on ice wi h ei he HLA-
A∗0201/TMFPHIIVDV pen ame o nega i e con ol pen ame ,
using 0.25 o 0.5 µg o he pen ame s pe condi ion. Cells we e
F on ie s in Immunology | www. on ie sin.o g 2No embe 2018 | Volume 9 | A icle 2782
Malm e al. Human No o i us T Cell Epi opes
u he s ained wi h monoclonal an ibodies agains human CD3
(clone UCHT1, luo escein iso hiocyana e (FITC) conjuga e)
and CD8a (clone RPA-T8, Pe CP Cy5.5 conjuga e) o 30 min
on ice. All eagen s used o s aining we e pu chased om
BD Pha mingen (San Jose, CA, Uni ed S a es). A e washing
he cells we e esuspended in 1% FBS, 2.5% o maldehyde in
PBS o low cy ome y acquisi ion. A leas 400 000 e en s
we e acqui ed o analysis on a 2-lase FACS Can oII low
cy ome e (BD) wi h FACSDi a So wa e V 6.1.3 (Bec on
Dickinson, Heidelbe g, Ge many). Da a we e analyzed by FlowJo
so wa e e sion 10.1 (T ee S a , San Ca los, CA, Uni ed S a es).
Lymphocy es we e ga ed acco ding o o wa d/sidewa d sca e
(FSC/SSC) and dead cells we e excluded by ga ing on
he popula ion nega i e o he iabili y dye. CD3+CD8+
popula ion was u he plo ed on SSC-A s. pen ame
popula ion.
ELISPOT IFN-γAssay
PBMCs o he ou dono s we e assayed in an ELISPOT
assay o IFN-γp oduc ion as p e iously desc ibed (17). The
cells we e s imula ed wi h inc easing concen a ions o 10-me
139TMFPHIIVDV148 pep ide (0.05, 0.1, 0.5, 1, 2, and 4 µg/ml
inal concen a ion), 4 µg/ml o 18-me pep ide 99-20, 4 µg/ml
i ele an 9-me pep ide (nega i e con ol) o 50 µg/ml o
phy ohemagglu inin (PHA, posi i e con ol). B ie ly, nine y-six-
well ni ocellulose il e pla es (Millipo e) we e coa ed wi h an i-
human IFN-γcap u e an ibody (Mab ech) and blocked wi h
10% FBS in CM. PBMCs (0.2 ×106cells/well) we e pla ed
wi h s imulan s and incuba ed o 20 h a +37◦C and 5% CO2.
Bio inyla ed an i-human IFN-γan ibody (Mab ech) ollowed
by s ep a idin-HRP (BD, New Je sey, Uni ed S a es) was used
o de ec ion. The spo s we e de eloped wi h Vec o No a
Red subs a e (Vec o Labs, Bu lingame, Uni ed S a es) and
he pla es we e analyzed using ImmunoSpo Se ies II analyze
(CTL Eu ope, Lein elden-Ech e dingen, Ge many). The esul s
a e exp essed as mean spo o ming cells (SFC)/106PBMCs o
he duplica e wells.
In acellula Cy okine S aining (ICS)
An ICS assay was employed o quan i y IFN-γ, TNF-α, and
IL-2 p oducing CD3+CD8+T cells. PBMCs we e s imula ed
acco ding o he p e iously published p o ocol (17) wi h 4 µg/ml
o 10-me pep ide 139TMFPHIIVDV148, 4 µg/ml i ele an
9-me pep ide (nega i e con ol) o 1 µg/ml S aphylococcal
en e o oxin B (SEB, Sigma) in he p esence o 1 µg/ml CD28 and
1µg/ml CD49d cos imula o y an ibodies (BD Biosciences, San
Jose, CA, Uni ed S a es). PBMCs incuba ed in CM supplemen ed
wi h he cos imula o y an ibodies only we e used as addi ional
con ol. The p o ein anspo inhibi o b e eldin A (GolgiPlug,
BD Biosciences, San Jose, CA, Uni ed S a es) a a concen a ion
o 10 µg/ml was added a e 2 h and he incuba ion was con inued
o 16 h a 37◦C. A e s imula ion, he cells we e ea ed
wi h EDTA o 15 min and washed wi h FACS S ain bu e .
P io o ixa ion and pe meabiliza ion o ICS, PBMCs we e
blocked o non-speci ic s aining, s ained o iable/non- iable
disc imina ion and su ace ma ke s CD3 and CD8a as desc ibed
abo e o he pen ame s aining. BD Fixa ion/Pe meabiliza ion
solu ion was used acco ding o he manu ac u e ’s ins uc ions
and cells we e in acellula ly s ained wi h he mix u e o IFN-γ
(clone 4S.B3) PE-Cy7 conjuga e, IL-2 (clone MQ1-17H12) PE-
conjuga e, and TNF-α(clone MAb11) allophycocyanin (APC)-
conjuga e in 50 µl Pe m/Wash bu e o 30 min on ice in
he da k. Cells we e esuspended in FACS S aining Bu e o
acquisi ion and analysis using FACS Can oII low cy ome e
and FACSDi a So wa e V 6.1.3. All eagen s used o ICS we e
pu chased om BD Pha mingen (San Jose, CA, Uni ed S a es).
The da a analysis was pe o med using FlowJo so wa e e sion
10.1.
RESULTS
The 10-me Minimal Epi ope P edic ion
and Conse a ion
A he ou se o ou s udy, an 18-me NoV VP1-speci ic CD8+T
cell epi ope (99-20, 134SPSQVTMFPHIIVDVRQL151) had been
iden i ied in wo subjec s, bo h ha ing HLA-A∗0201 allele. When
analyzed by IEDB da abase ANN me hod, a 10 aa sequence
139TMFPHIIVDV148 wi hin he 18-me showed he highes
binding a ini y o IC50 21.5 nM (Table 1), whe eas all o he
p edic ed sequence leng hs indica ed a ini y >80 nM (da a no
shown). Pep ides wi h IC50 <50 nM a e conside ed high a ini y
and <500 nM in e media e a ini y (30). Cong uen ly, he same
10-me epi ope was iden i ied o ha e he highes binding a ini y
by SMM me hod [da a no shown (23)]. SYFPEITHI p edic ion
algo i hm con i med a sco e o 27 o he pep ide a ini y o
HLA-A∗0201 allele (Table 1). When compa ing he aa sequence
o di e en NoV geno ypes (Table 1) he 10-me epi ope was
ound o be highly conse ed among di e en GII.4 a ian s
and also mo e dis an NoV geno ypes belonging o GII and
GI. The phylogene ic dis ances o he NoV geno ypes compa ed
in he Table 1 a e shown in Figu e 1. In addi ion, he 10-me
epi ope sequence in all geno ypes belonging o GI and GII NoV
lis ed in he Table 1 also had good a ini y binding sco es o
HLA-A∗0201 allele ega dless o up o h ee aa subs i u ions
compa ed o he GII.4-1999 10-me sequence. On he con a y,
he 10-me sequence in he human GIV 2010 NoV had a
unique subs i u ion 140M o 140Q which seemed o abolish i s
binding a ini y esul ing in IC50 552.6 nM and sy pei hi sco e
17 (Table 1). In addi ion, a change o he aa 148V o 148D a
he c- e minus o he empi ical 10-me pep ide abolished he
p edic ed binding comple ely. This is no su p ising as he aa
a posi ions 140 and 148 a e epo ed ancho ing posi ions o
HLA-A∗0201 binding (31–33). In addi ion, dele ions a he each
e minus o he pep ide a ec ed he p edic ed binding capabili y
o he pep ide.
CD8+T Lymphocy es Bind he 10-me
Pep ide 139TMFPHIIVDV148 in he Con ex
o HLA-A∗0201
The equency o T cells speci ic o 139TMFPHIIVDV148
10-me in he con ex o HLA-A∗0201 allele was analyzed
by di ec binding o pen ame A∗0201/TMFPHIIVDV R-PE
F on ie s in Immunology | www. on ie sin.o g 3No embe 2018 | Volume 9 | A icle 2782
Malm e al. Human No o i us T Cell Epi opes
TABLE 1 | Conse a ion and HLA-A2*0201 allele binding a ini y p edic ion o he no o i us 10-me epi ope.
No o i us Geno ype Accession numbe 10-me aa sequence
(aa 139–148)
Iden i y % IC50 nM (ANN)aSy pei hi sco eb
GII.4-1999 AAD40490.1 TMFPHIIVDV100 21.5 27
GII.4 Fa ming on Hills 2002 AFJ04708.1 TMFPHIIVDV 100 21.5 27
GII.4 Apeldoo n 2003 BAF74517.1 TMFPHIIVDV 100 21.5 27
GII.4 Hun e 2004 AAZ31376.2 TMFPHIIVDV 100 21.5 27
GII.4 New O leans 2009 ADD10375.1 TMFPHIIVDV 100 21.5 27
GII.4 Sydney 2012 AFV08795.1 TMFPHIVVDV 90 28.22 27
GII.17 Kawasaki308 BAR42289.1 TMLPHLIVDV 80 30.14 28
GI.3 2002 AAL12962.1 TMFPHVIADV 80 14.44 27
GII.12 Wo ley 1990 CAB89099.1 TMFPHVIIDV 80 31.84 25
GII.10 Vie nam026 2005 AAT12445.1 TMFPHVIIDV 80 31.84 25
GII.1 1971 AFS33555.1 TMFPHVIIDV 80 31.84 25
GII.2 2014 BAV19452.1 TMFPHVIIDV 80 31.84 25
GII.3 1976 AED02039.1 TMCPHVIVDV 80 278.56 26
GI.1 Wes Ches e 2001 AAS86807.1 TLFPHVIADV 70 16.87 29
Human GIV 2010 AFJ21376.1 TQFPHVIIDV 70 552.6 17
No exis ing TMFPHIIVDD 90 17260.44 17
9-me _MFPHIIVDV 90 4645.06 16
9-me TMFPHIIVD_ 90 9837.8 15
aInhibi o y concen a ion (IC)50 alues we e ob ained using IEDB analysis esou ce ANN aka Ne MHC ( e . 4.0) ool.
bSy pei hi sco e was de i ed om he Epi ope P edic ion ool by h p://www.sy pei hi.de/ Bolded le e s indica e he epo ed ancho esidues o HLA-A2*0201 allele binding epi opes
(31,32). Unde lined le e s indica e he aa changes wi h espec o GII.4-1999-de i ed 10-me epi ope. I alic on deno es sequences wi h no a ini y binding o HLA-A2*0201.
FIGURE 1 | Phylogene ic analysis o no o i us majo capsid p o ein VP1
amino acid sequences. The ee was in e ed by Neighbo -Joining me hod
wi h a boo s ap o 500 by using MegaX. The scale ba shows he gene ic
dis ance, exp essed as amino acid subs i u ions pe si e.
using ou -colo low cy ome y. PBMCs o he wo HLA-
A∗0201 posi i e dono s p e iously esponding o he 18-me
pep ide 99-20 had equency o 0.51% (Dono 1) and 1.52%
(Dono 2) (Figu e 2) pen ame -binding T cells o he li e
CD3+CD8+ga ed lymphocy es. A he same ime, he CD8+
T cells o he con ol dono s non- esponde s o he 99-20, did
no bind o A∗0201/TMFPHIIVDV pen ame (Figu e 2). The
nega i e con ol pen ame did no show non-speci ic binding
(Figu e 2).
The 10-me 139TMFPHIIVDV148 Epi ope
Induces S ong IFN-γResponse in
HLA-A∗0201 Posi i e Dono s
IFN-γ-sec e ing lymphocy es speci ic o 139TMFPHIIVDV148
10-me pep ide we e enume a ed by ex- i o ELISPOT assay
wi h inc easing concen a ion o he 10-me pep ide. Robus
IFN-γ esponse o he 10-me pep ide was obse ed o he
wo HLA-A∗0201 posi i e dono s (Figu e 3) bu no o he
wo con ol dono s (da a no shown). A dose esponse was
obse ed up o 1.0 µg/ml concen a ion o he pep ide and
also e y low concen a ion (0.05 µg/ml) s imula ed high IFN-
γsec e ion (dono 1, 260 SFC/106cells; dono 2, 293 SFC/106
cells). IFN-γp oduc ion was no obse ed in he cells s imula ed
wi h he i ele an 9-me pep ide o CM only (SFC/106cells
<50) (Figu e 3A–C). All dono s esponded s ongly o he
posi i e con ol PHA (IFN-γSFC/106cells >2000, da a no
shown).
The 10-me 139TMFPHIIVDV148 Epi ope
Induces Mul i unc ional CD8+T Cells
P oducing IFN-γ, IL-2, and TNF-α
Along wi h quan i ica ion o he epi ope-speci ic CD8+T cells
wi h HLA-A∗0201 pen ame and IFN-γELISPOT, immune
esponses o 10-me pep ide we e cha ac e ized by ICS o
F on ie s in Immunology | www. on ie sin.o g 4No embe 2018 | Volume 9 | A icle 2782
Malm e al. Human No o i us T Cell Epi opes
FIGURE 2 | Pen ame s aining o no o i us-de i ed 139TMFPHIIVDV148 epi ope-speci ic CD8+T cells. Pe iphe al blood mononuclea cells (PBMCs) om wo
HLA-A*0201-posi i e heal hy adul dono s and he nega i e con ol we e di ec ly s ained using 0.25 and 0.5 µg HLA-A*0201/TMFPHIIVDV pen ame o nega i e
con ol pen ame . The do plo s show he pe cen age o pen ame -posi i e T cells o wo HLA-A*0201-posi i e dono s and a ep esen a i e nega i e con ol dono
a e ga ing on li e CD3+CD8+T lymphocy e popula ion.
IFN-γ, IL-2, and TNF-α(Figu e 4). The ga ing s a egy o
ICS analysis is shown in Figu e 4. The iabili y o he
lymphocy es was >98% and app oxima ely 30% o he iable
CD3+lymphocy e popula ion we e CD8+T cells. CD8+T cells
we e u he seg ega ed in o IFN-γ+and IFN-γ−popula ion
and plo ed o he exp ession o IL-2 and TNF-α. Robus
cy okine esponse by CD3+CD8+T cells owa d 10-me
epi ope in dono 1 (Figu e 4A) and dono 2 (Figu e 4B) bu
no in wo con ol dono s (da a no shown) was obse ed.
Bo h dono s had CD8+T cells p oducing single, double,
and iple cy okines (Figu es 4A,B). App. 1% o he CD8+
T cells o bo h dono s p oduced IFN-γin esponse o he
10-me epi ope (Figu e 4) whe eas i ele an con ol pep ide
induced no IFN-γ(<0.05%, da a no shown). The 10-me
epi ope induced mul i unc ional CD8+T cells in bo h dono s,
dono 1 had ∼30% iple cy okine (IFN-γ+, TNF-α+, and IL-
2+) sec e ing CD3+CD8+T cells, while dono 2 had ∼60%
o he iple-posi i e CD8+T cells. S ong IFN-γ, IL-2, and
TNF-α esponse was obse ed in bo h CD8+and CD8−
PBMCs agains posi i e con ol SEB in all es ed dono s (no
shown).
DISCUSSION
Despi e signi ican e o s o de ine co ela es o p o ec ion o
NoV in ec ion (1,2), he ole o T lymphocy es in p o ec ion
and clea ance o NoV in ec ion is s ill la gely unexplo ed a ea,
wi h only ew pape s published so a (16–19,34). We ha e
ecen ly es ed en heal hy olun ee dono s o NoV GII.4
capsid VP1-speci ic T cell esponses using ma ix pep ide pools
and ound wo CD8+T cell esponde s o 18-me NoV-
speci ic pep ide (134SPSQVTMFPHIIVDVRQL151, 99-20) (17).
In he e, we de ined a 10-me 139TMFPHIIVDV148 minimal
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Malm e al. Human No o i us T Cell Epi opes
FIGURE 3 | No o i us epi ope 139TMFPHIIVDV148 –speci ic enzyme-linked immunoso ben spo (ELISPOT) in e e on-gamma (IFN-γ) esponses. PBMCs o Dono 1
(A,B) and Dono 2 (A,C) posi i e o HLA-A*0201 allele we e s imula ed wi h an inc easing concen a ion (0–4 µg/ml) o he 10-me epi ope pep ide, 4 µg/ml i ele an
9-me pep ide (C l), o le uns imula ed (CM, cul u e media). Images o he ELISPOT eplica e wells wi h he s ained spo - o ming cells (SFC) a e shown (A). Mean
SFC/106PBMCs o wo eplica e wells wi h he s anda d e o s o he mean o Dono 1 (B) and Dono 2 (C). The cu -o line (do ed line) indica es he posi i e
SFC/106 alue o ≥50 SFC/106cells and wice abo e he backg ound con ol (CM only wells).
epi ope wi hin his sequence and i s es ic ion o HLA-A∗0201
allele.
While he 10-me 139TMFPHIIVDV148 epi ope is de i ed
om GII.4-1999 s ain (17), he alignmen wi h se e al
o he GII.4 a ian s showed epi ope sequence o emain
in a iable un il 2012 (Table 1). In e es ingly, alignmen wi h
o he qui e dis an geno ypes (Figu e 1) e en belonging o GI
i uses, indica ed high deg ee o conse a ion o he epi ope.
Impo an ly, up o h ee aa subs i u ions as in he GI.1 Wes
Ches e 2001 NoV do no necessa ily educe he binding
a ini y o HLA-A∗0201, depending on he aa posi ion and
cha ac e is ics. Aa a he posi ion 6 and 8 o he 10-me
showed mos a iabili y among he aligned geno ypes, howe e ,
hese changes did no lead o conside able change in binding
a ini y p edic ions. Howe e , a change a aa posi ion 2 om a
hyd ophobic (M) o a pola aa (Q) in a human GIV 2010 NoV
and a aa posi ion 9 om V o D in an empi ical sequence seemed
o d as ically abolish binding a ini y. This is no su p ising as
HLA-A∗0201 binding epi opes ha e a es ic ed size o 9/10 aa
and hyd ophobic ancho esidues a posi ions 2 and 9/10 likewise
he epi ope iden i ied in his s udy. The NoV 10-me epi ope
con ains me hionine (M) a posi ion 2 and aline (V) a posi ion
10 which is desc ibed as a ypical C- e minal ancho esidue
(31–33).
Di ec s aining o PBMCs wi h HLA-A∗0201 pen ame
loaded wi h 139TMFPHIIVDV148 pep ide a es ed ela i ely high
equency o ci cula ing NoV capsid-speci ic memo y CD8+
T cells in wo HLA-A∗0201 posi i e heal hy dono s (0.51 and
1.52% o CD3+CD8+ga ed lymphocy es, espec i ely). HLA-A2
is he mos p e alen MHC allele amily in human popula ion
and he gene equency o HLA-A∗02:01 is especially high in
Caucasians (>20%), and o he wes e n e hnic g oups (A ican
Ame icans 12%, Hispanics 23%, No h Ame ican na i es 21%)
(35–37), u he emphasizing he impo ance o he iden i ied
epi ope. Howe e , i has been epo ed ha only a small po ion
o he pep ides wi h p edic ed and expe imen ally con i med
high a ini y binding, a e ac ually capable o induce T-cell
esponses (38). The e o e, he u iliza ion o unc ional assays is
essen ial o de e mining he immunogenici y o an epi ope. To
ha end, he unc ionali y o T cell immune esponses o 10-me
epi ope we e de e mined by wo unc ional assays, ELISPOT
IFN-γand ICS. The esul s o ELISPOT IFN-γassay showed
obus NoV VP1 10-me epi ope-speci ic CD8+T cell immune
esponses in HLA-A∗0201-posi i e indi iduals, whe eas con ol
dono s we e nega i e. Ti a ion o 10-me pep ide down o
0.05 µg/ml e ealed high a idi y o CD8+T cells o 10-me
pep ide. These esul s we e expanded using ICS assay o de ec
CD8+T cells sec e ing cy okines IFN-γ, IL-2, and TNF–α. By
his app oach, he p esence o ci cula ing mono unc ional and
poly unc ional memo y CD8+T lymphocy es speci ic o 10-
me NoV epi ope was de ec ed in HLA-A∗0201-posi i e heal hy
dono s. Poly unc ional CD8+T cells ha p oduce mo e han
one immune media o , a e associa ed wi h p o ec ion om i al
in ec ions, such as HIV-1 and human he pes i us (39–41). In
he u u e we aim o u ilize he pen ame echnology o alida e
CD8+T cell esponses in a la ge numbe o se ologically
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Malm e al. Human No o i us T Cell Epi opes
FIGURE 4 | Func ional cha ac e iza ion o no o i us 10-me epi ope 139TMFPHIIVDV148 –speci ic CD8+T cells by in acellula cy okine s aining (ICS). P oduc ion o
IL-2, IFN-γ, and TNF-αmeasu ed by ICS a e 16h s imula ion o he PBMC wi h he pep ide 139TMFPHIIVDV148 (4 µg/ml). Shown a e he esul s o he wo
HLA-A*0201-posi i e dono s, Dono 1 (A) and Dono 2 (B). Li e epi ope-speci ic CD3+CD8+T cells we e seg ega ed in o IFN-γ+and IFN-γ−T cells. Se en dis inc
popula ions, based on p oduc ion o he cy okines in any combina ion a e depic ed on he pie cha s and he accompanying ables. F ac ions o he cells p oducing
one, wo, o h ee cy okines a e de i ed om he plo ed IFN-γ+and IFN-γ−popula ions.
posi i e NoV-in ec ed HLA-A∗02:01 posi i e subjec s o
s eng hen he clinical signi icance o he inding in his
s udy.
Pen ame s aining, ELISPOT IFN-γand ICS assays,
pe o med simul aneously using he same sample o each
dono , esul ed in highly cong uen da a suppo ing high
immunogenici y o he no el NoV 139TMFPHIIVDV148 CD8+
T cell epi ope. Many i al in ec ions a e no con ained by
an ibody esponses alone and i is likely ha T cell esponses
play a ole in clea ance o NoV in ec ion and may ha e a ole in
p o ec i e immuni y as well. Vi us-speci ic CTL esponses play a
c ucial ole in a clea ance o many o he i al in ec ions such as
human immunode iciency i us-1 (HIV-1) (40,42,43), in luenza
(22,44) and human papilloma i us (45). Pa ien s wi h ch onic
hepa i is B (HBV) in ec ion ypically lack e ec i e HBV-speci ic
T cells, whe eas ully eco e ed pa ien s display s ong CD8+
T-cell esponses (46).
To conclude, he p esen wo k desc ibes he i s human
NoV CD8+T cell epi ope 139TMFPHIIVDV148 es ic ed o
HLA-A∗02:01 allele. Rega ding he high conse a ion o he
iden i ied epi ope and high equency o HLA-A∗02:01 allele
in human popula ion, i can be specula ed ha ∼20% o
he indi iduals exposed o di e gen NoV geno ypes, will
de elop s ong CD8+T cell esponses o his 10-me epi ope.
While NoV blocking an ibodies a e la gely geno ype speci ic,
a ole o T cells a ge ed o b oadly conse ed epi opes, as
desc ibed in he e, may be o la ge signi icance. Howe e , he
ole and impo ance o his epi ope as well as o e all T cell
esponses in p o ec ion om NoV in ec ion needs o be u he
in es iga ed.
AUTHOR CONTRIBUTIONS
MM sample acquisi ion, p ocessing, and labo a o y analysis
(Pen ame s aining, ELISPOT, and ICS by low cy ome y).
Da a acquisi ion, analysis, and in e p e a ion, and w i ing he
manusc ip . TV he Head o Vaccine Resea ch Cen e , e ision o
he manusc ip ex . VB he Head o he Labo a o y, concep ion
and designing he s udy, da a in e p e a ion, d a ing, and w i ing
he pape , c i ical e ision o a icle o impo an in ellec ual
con en .
F on ie s in Immunology | www. on ie sin.o g 7No embe 2018 | Volume 9 | A icle 2782
Malm e al. Human No o i us T Cell Epi opes
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.
ACKNOWLEDGMENTS
We g a e ully acknowledge he echnical assis ance gi en by
he labo a o y pe sonnel o he Vaccine Resea ch Cen e o
Uni e si y o Tampe e.
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