O iginal a icle
De elopmen and ma u a ion o no o i us an ibodies in childhood
Vesna Blaze ic
a
, Ma ia Malm
a,
*, Hanna Honkanen
b
, Mikael Knip
c,d,e,
, Heikki Hy€
o y
b,g
,
Timo Vesika i
a
a
Vaccine Resea ch Cen e , Uni e si y o Tampe e Medical School, Tampe e, Finland
b
Depa men o Vi ology, Uni e si y o Tampe e Medical School, Tampe e, Finland
c
Tampe e Cen e o Child Heal h Resea ch, Tampe e Uni e si y Hospi al, Tampe e, Finland
d
Child en's Hospi al, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki, Finland
e
Resea ch P og ams Uni , Diabe es and Obesi y, Uni e si y o Helsinki, Helsinki, Finland
Folkh€
alsan Resea ch Cen e, Helsinki, Uni e si y o Helsinki, Finland
g
Fimlab Labo a o ies, Pi kanmaa Hospi al Dis ic , Tampe e, Finland
Recei ed 28 Augus 2015; accep ed 15 Decembe 2015
A ailable online 25 Decembe 2015
Abs ac
The bu den o no o i us (NoV) gas oen e i is is subs an ial in young child en. Ma e nal an ibodies a e hough o p o ec a child om NoV
in ec ion in ea ly in ancy bu subsequen de elopmen o NoV-speci ic p o ec i e immuni y in child en is s ill la gely unexplo ed.
We ha e de e mined NoV-speci ic an ibody se ocon e sion o GII.4 i us-like pa icles as an indica o o NoV in ec ion in wo child en
p ospec i ely ollowed om bi h o eigh yea s o age. Blocking ac i i y and a ini y ma u a ion o ma e nal and se um IgG an ibodies we e
e alua ed.
Ou esul s show ha mul iple in ec ions occu in child en up o eigh yea s o age. The i e , blocking ac i i y and a idi y o ma e nal
an ibodies de e mined suscep ibili y o an in an o NoV in ec ion. NoV GII.4-speci ic an ibodies wi h high blocking po en ial and a idi y we e
de eloped a wo o h ee yea s o age and we e e ained h oughou he ollow-up. Subsequen NoV in ec ions may ha e con ibu ed o he
du a ion o p o ec i e NoV-speci ic immune esponses ha las ed o se e al yea s.
This s udy adds o cu en unde s anding o he du a ion o passi e p o ec ion by ma e nal an ibodies and he du a ion and quali y o acqui ed
immuni y ollowing p ima y and subsequen NoV in ec ions in in an s and young child en, who a e he main a ge g oup o NoV accine
de elopmen .
©2016 The Au ho s. Published by Else ie Masson SAS on behal o Ins i u Pas eu . 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/).
Keywo ds: No o i us; In an ; Child en; Immuni y; In ec ions; Se ology
1. In oduc ion
In an s and child en unde i e yea s o age a e mos sus-
cep ible o no o i us (NoV) acu e gas oen e i is (AGE)
[1e3], and a e he e o e one o he po en ial a ge g oups o
NoV accina ion. Young child en ha e highe and longe i al
shedding compa ed o adul s [4] and may be conside ed as a
pool o ansmission o NoV o o he ulne able popula ions,
e.g. senio ci izens. NoV in ec s all age g oups and in adul s
he NoV se oposi i i y is nea 100% as a esul o li e ime
NoV exposu e his o y [5].
Mos NoVs in ec ing humans belong o genog oups GI and
GII, which a e gene ically so dis an ha no in e genog oup
c oss-p o ec i e immune esponses a e gene a ed [6].How-
e e , a e na u al in ec ion [7e11] and also accina ion [12]
a iable deg ee o in agenog oup c oss- eac i i y has been
obse ed. NoV GII.4 geno ype is esponsible o
*Co esponding au ho . Vaccine Resea ch Cen e , Uni e si y o Tampe e
Medical School, Bioka u 10, FI-33520 Tampe e, Finland. Tel.: þ358 50 318
6882; ax: þ358 3 364 1512.
E-mail add ess: [email p o ec ed] (M. Malm).
Mic obes and In ec ion 18 (2016) 263e269
www.else ie .com/loca e/micin
h p://dx.doi.o g/10.1016/j.micin .2015.12.004
1286-4579/©2016 The Au ho s. Published by Else ie Masson SAS on behal o Ins i u Pas eu . 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/).
app oxima ely 55e80% o all NoV in ec ions wo ldwide
[13e15]. New epidemic an igenically dis inc GII.4 a ian s
eme ge e e y wo o h ee yea s [16e18] bu conside able
an ibody c oss- eac i i y be ween GII.4 a ian s has been
obse ed e en in young child en [19].
Co ela es o p o ec i e immuni y o NoV a e no well
es ablished. A common inding has been ha p o ec ion is no
long las ing [5]. Ea ly homologous challenge s udies by Pa -
ino e al. [20] and Johnson e al. [21] ha e shown ha
du a ion o p o ec i e immuni y a ies om wo mon hs o
wo yea s. The e a e a limi ed numbe o s udies on NoV-
speci ic immune esponses in child en. In con as o hose
o adul s, IgG esponses o NoV du ing he i s yea o li e a e
ela i ely weak and sho -li ed and he an ibodies a e o a low
a idi y [22,23]. Sai o e al. [24] ound ha mos in ec ions in
less han six-mon h-old in an s a e asymp oma ic, p obably
due o ma e nally acqui ed an ibodies and, possibly, b eas -
eeding. Al oge he , he build-up o p o ec i e immuni y a e
p ima y and seconda y NoV in ec ions is no well
cha ac e ized.
NoV i us-like pa icles (VLPs) a e commonly used o
s udying immune esponses agains NoV. The exp ession o
he NoV capsid VP1 p o ein esul s in o ma ion o VLPs ha
a e mo phologically and an igenically simila o na i e i ions
[25], he eby being also p omising accine candida es [26,27].
Blocking assay has been used as a su oga e measu e o NoV
neu aliza ion, de e mining he abili y o se um an ibodies o
block binding o NoV VLP o i s pu a i e his o-blood g oup
an igen (HBGA) ecep o s/a achmen ac o s [12,28,29].We
ha e p e iously used NoV GII.4 VLPs o de e mine NoV
se op e alence in Finnish child en [10]. Also, mo e ecen ly
an endpoin i e >51,200 and/o 90% blocking i e (BT
90
)
>100 was sugges ed as an indica o o p o ec ion om NoV
in ec ion in hese child en [10]. The ecen challenge s udies
in adul s ha e u he a i med co ela ion be ween he
blocking an ibodies and p o ec ion [12,30]. An ibody a idi y
is low in p ima y and ecen NoV in ec ions and inc eases wi h
ime [23], bu he ele ance o he Ab a idi y in NoV p o-
ec i e immuni y is no cu en ly known.
In his s udy we de e mined se ocon e sion o NoV GII.4
VLPs as an indica o o NoV in ec ion in wo child en p o-
spec i ely ollowed om bi h up o he age o eigh yea s and
examined he du a ion, blocking abili y, and a idi y o he
an ibodies.
2. Ma e ials and me hods
2.1. S udy samples
Two heal hy child en (Subjec 1 and 2) aking pa in he
Type I Diabe es P edic ion and P e en ion (DIPP) s udy [31]
we e p ospec i ely ollowed o NoV GII.4-speci ic an i-
bodies om bi h o eigh yea s o age. The DIPP s udy
p o ocol was app o ed by he e hics commi ee o he Pi -
kanmaa Hospi al Dis ic (Pe mi numbe : 97193M) and a
w i en in o med consen was ob ained om he pa en s.
Heal h eco ds we e collec ed du ing he ollow-up isi s
and symp oms ela ed o acu e gas oen e i is episodes we e
eco ded. Subjec 1 had been b eas ed exclusi ely o i s
i e mon hs and pa ially un il he age o 25 mon hs.
B eas eeding o he Subjec 2 was exclusi e o i s wo
mon hs, pa ial o one mon h. All p ocedu es pe o med
we e conduc ed acco ding o he p inciples exp essed in he
Decla a ion o Helsinki. Co d blood samples we e aken a
bi h in yea 2000 and he ea e blood samples we e aken
in sodium ci a e ubes a he age o 3, 6, 12, 17 and 24
mon hs and he ea e once pe yea up o eigh yea s o age.
Plasma ac ion was s o ed a 70 C un il analyzed. Non-
dia heal s ool samples we e collec ed mon hly a he age
o 3e9 mon hs and e e y wo mon hs un il he age o 1 yea ,
9 mon hs. Vi al RNA was ex ac ed om he s ool suspen-
sions and s o ed a 70 C un il e e se ansc ip ion-
polyme ase chain eac ion (RT-PCR) and open eading
ame 1 polyme ase ( egion A) sequencing we e used o
NoV geno yping acco ding o p e iously desc ibed me hods
[32].
2.2. No o i us VLP p oduc ion
GII.4-1999 capsid VP1 sequence o igina ed om a pa ien
sample collec ed in 1999 (GenBank e e ence s ain accession
no. AF080551) [23,33]. NoV GII.4-1999 VLPs used as an i-
gens in analy ical me hods we e p oduced by a baculo i us
exp ession sys em (In i ogen) in Spodop e a ugipe da (S 9)
insec cell cul u es. VLPs we e pu i ied wice wi h discon-
inuous suc ose g adien ul acen i uga ion as p e iously
desc ibed [33]. The o al p o ein concen a ion was quan i ied
wi h Pie ce
®
BCA P o ein Assay (The mo Scien i ic, Rock-
o d). P o ein pu i y, in eg i y, and mo phology we e de e -
mined by 12% sodium dodecyl sul a e polyac ylamide gel
elec opho esis (SDS-PAGE), wes e n blo ing and ans-
mission elec on mic oscopy (EM) as desc ibed ea lie
[26,33].
2.3. Se um IgG ELISA
NoV GII.4-1999-speci ic IgG an ibody le els we e
analyzed by ELISA as ea lie desc ibed [10]. Se um speci-
mens we e dilu ed wo- old s a ing a 1:100 and pla ed on
GII.4-1999 VLP coa ed (0.5 mg/ml in phospha e-bu e ed sa-
line, PBS) 96-well hal -a ea mic o i e pla es (Co ning Inc.,
Co ning, NY) blocked wi h 5% skimmed milk in PBS. Se um
dilu ions we e incuba ed on pla es o 1 h a 37 C. Bound
GII.4-1999-speci ic an ibodies we e de ec ed wi h goa an i-
human IgG-HRP (In i ogen, CA, USA) ollowed by o-phe-
nylenediamine (OPD) subs a e (SigmaeAld ich, MO, USA)
and H
2
O
2
. Op ical densi y (OD) was measu ed a l490 nm
using he Vic o 2 1420 Mul ilabel Coun e (Wallac, Pe kin
Elme ) pla e eade . Backg ound signal om he blank wells
(wells wi hou se um) was sub ac ed om all o he OD
eadings on he pla e. Each pla e con ained NoV nega i e and
posi i e con ol se um sample as an assay con ol. The cu -o
alue was de e mined as he mean OD eading o he nega i e
con ol se um wells a a dilu ion 1:200 þ3s anda d e o
264 V. Blaze ic e al. / Mic obes and In ec ion 18 (2016) 263e269
and a leas 0.100 OD. Endpoin i e was exp essed as a
ecip ocal o he inal se um dilu ion gi ing an OD abo e he
cu -o alue. Se ocon e sion was de ined as a leas ou - old
inc ease in he i e o successi e se a.
2.4. A idi y assay
A idi y o se um IgG an ibodies was de e mined in
ELISA by u ea elu ion s eps acco ding o a p e iously pub-
lished me hod [23,34]. Se a we e es ed a 1:100 dilu ion on
GII.4-1999 VLP (1.0 mg/ml) coa ed and blocked mic o i e
pla es. A e incuba ion o 1 h a 37 C, wells we e exposed
wice o 5 min o 8 M u ea in PBS-Tween solu ion o o
phospha e bu e . A e washing, he wells we e incuba ed
wi h pe oxidase-labeled an i-human IgG and bound an i-
bodies we e de ec ed as desc ibed abo e. A idi y index (%)
was calcula ed using he equa ion: [OD wi h u ea/OD
wi hou u ea] 100%. An a idi y index >50% was consid-
e ed as high a idi y [7,23].
2.5. Blocking o HBGA binding
The abili y o an ibodies o block GII.4-1999 VLP binding
o syn he ic HBGA was es ed using H ype 1 HBGA ca -
bohyd a es ha ha e been shown o be biologically ele an
o NoV a achmen in in ec ion [28] as well as o bind o
GII.4 NoV VLPs [35]. The blocking assay was ca ied ou as
p e iously desc ibed [35]. B ie ly, he p e-coa ed and p e-
blocked Neu A idin pla es (Pie ce, Rock o d, IL) we e
coa ed wi h H ype 1 (Glyco ech, Gai he sbu g, MD) HBGAs
o 1 h a oom empe a u e. Se ially wo- old dilu ed se um
samples s a ing a 1:50 dilu ion we e i s p eincuba ed wi h
NoV GII.4-1999 VLPs (0.4 mg/ml) o 1 h a 37 C, be o e
incuba ing he samples on HBGA-coa ed Neu A idin pla es
o 2ha þ4C. Bound VLPs we e de ec ed by GII.4-1999-
speci ic mouse an ise a and an i-mouse IgG-HRP (Sigma-
eAld ich, Sain Louis, MO), ollowed by OPD subs a e and
H
2
O
2
. Mean OD eading o he blank wells was sub ac ed
om he OD alues o he sample wells. A maximum binding
signal was he mean OD o he wells wi h VLP alone, lacking
se um. The blocking index was de ined as ollows: 100 e
[OD o wells wi h se um/OD o wells wi hou
se um] 100. A blocking i e (BT) alue o 50 o 90 was
de e mined as he ecip ocal o he inal se um dilu ion ha
blocked a leas 50% o 90% o VLPs binding o he HBGA.
Fo s a is ical analyses an a bi a y BT o 25 (hal o he
s a ing ecip ocal se um i e 50) was assigned o all se a
which lacked blocking.
2.6. S a is ical analysis
The Spea man ank co ela ion coe icien was used o
examine he di e ences be ween an ibody i e s, blocking i-
e s BT
90
and a idi y index. All hypo hesis es ing was wo-
ailed. S a is ical analyses we e pe o med using IBM SPSS
S a is ics (SPSS, Chicago, IL) e sion 22.0. P <0.05 was
conside ed s a is ically signi ican .
3. Resul s
The in eg i y and mo phology o NoV GII.4 VLPs used o
he analy ical me hods we e e i ied by wes e n blo (Fig. 1A)
and EM (Fig. 1B). EM images iden i ied NoV VP1 capsid
p o eins sel -assembled in o he VLPs o ~38 nm in size.
The Subjec 1 had mode a e le els (endpoin i e 3200) o
GII.4-1999-speci ic co d blood an ibodies ha waned in he
ollowing h ee mon hs (Fig. 2 and Table 1). Se ocon e sion o
NoV GII.4 VLPs was obse ed a he age o six mon hs and
again a he h ee and i e yea s o age, indica ing a leas h ee
NoV in ec ions by his age. No NoVs we e de ec ed in he
s ool samples o he Subjec 1 collec ed du ing he i s wo
yea s o li e. Only one episode o AGE be ween he age o
17e24 mon hs had been eco ded o Subjec 1, which did no
coincidence wi h he s ool o se um samples collec ed.
Despi e he se ocon e sion, he p ima y NoV exposu e
be o e he age o six mon hs did no inc ease he blocking
(BT
90
<50) o a idi y index (19%) o GII.4-1999-speci ic
an ibodies (Table 1). Blocking an ibodies wi h BT
90
alue o
100 and high a idi y (95% a idi y index) we e obse ed a e
he p esumably second NoV in ec ion a he age o h ee yea s.
The abili y o block he binding o VLPs o HBGA ligand
declined wi hin one yea (BT
90
<50), bu emained s ill abo e
he ini ial le el un il he age o se en yea s (BT
50
50). Simi-
la ly, he a idi y index declined wi hin one yea o 67%, bu
emained >50% o all he subsequen ime poin s. A wo- old
inc ease in endpoin i e was de ec ed again a he age o eigh
yea s bu no change in blocking o a idi y index was obse ed.
In compa ison o he Subjec 1, he second child (Subjec 2)
had conside ably highe le els o GII.4-1999-speci ic co d
blood an ibodies (endpoin i e 51,200) wi h BT
90
100 and
a idi y index 95%, which d opped by he age o six mon hs
(Fig. 3 and Table 2). The p ima y NoV in ec ion was acqui ed
be ween 1.5 and 2 yea s o age (Table 2), app oxima ely a yea
la e han in he Subjec 1 (Table 1). In addi ion, his in ec ion
induced highly unc ional (BT
90
100 and a idi y index 99%)
GII.4-1999-speci ic an ibodies. NoV RT-PCR and sequencing
o he s ool samples de ec ed NoV GII.2 geno ype in ec ion a
he age o 1 yea and 9 mon hs, p io o he i s se ocon e -
sion o NoV GII.4 VLPs a he age o wo yea s (Table 2 and
Fig. 3). Co espondingly, an episode o AGE had been
eco ded be ween he age o 17e24 mon hs. Despi e he high
le els o p esumably p o ec i e an ibodies a he age o wo
yea s, ano he NoV in ec ion al eady appea ed du ing he nex
yea as indica ed by he ou - old inc ease in BT
90
a he age
o h ee. Judging by he se ocon e sion, he Subjec 2 acqui ed
wo addi ional NoV in ec ions a he ages o i e and eigh
yea s (Table 2). Addi ionally, he Subjec 2 had AGE symp-
oms a he age o 4.5 yea s, co esponding o se ocon e sion
a he age o 5 yea s.
Al oge he , bo h subjec s acqui ed a leas h ee o ou
NoV in ec ions (Tables 1 and 2). The Subjec 1 who had low
ma e nal an ibody i e , lacking blocking ac i i y and a idi y,
acqui ed i s in ec ion al eady by he age o six mon hs,
whe eas he Subjec 2, wi h a high i e and unc ionali y o
ma e nal an ibodies acqui ed i s in ec ion a leas one yea
265V. Blaze ic e al. / Mic obes and In ec ion 18 (2016) 263e269
la e . An ibodies wi h high blocking ac i i y and a idi y we e
de eloped only a (Subjec 2) o a e (Subjec 1) he wo yea s
o age. Al hough he a idi y emained high up o eigh yea s
o bo h subjec s, only he Subjec 2 e ained high le el o
blocking an ibodies. Howe e , hese GII.4-speci ic an ibodies
did no p o ec he Subjec 2 om a new in ec ion p io o he
age o eigh yea s wi h p esumably NoV geno ype highly
di e gen o GII.4. Al oge he , a posi i e co ela ion was
de ec ed be ween BT
90
and IgG endpoin i e s ( ¼0.808,
P<0.01) and a idi y index ( ¼0.775, P <0.01) o se um
an ibodies in bo h subjec s.
4. Discussion
In he p esen s udy we ollowed he de elopmen o NoV
GII.4-speci ic humo al immune esponses in wo child en
om bi h up o eigh yea s o age. Se ocon e sion was
de ec ed in a leas h ee ins ances, sugges ing a minimum o
h ee NoV GII in ec ions in bo h child en in eigh yea s. NoV
GII.4 VLPs we e chosen o measu e an ibody esponses o GII
NoVs as i is he mos common geno ype ci cula ing o >20
yea s and causing spo adic acu e gas oen e i is in child en
wo ldwide [15,18,36]. Also, mos in ec ions in child en du ing
he yea s 2000e2013 in Finland we e caused by he GII.4
i uses [14,37].
In he e, he ou ine s ool specimens collec ed p io o wo
yea s o age we e used o de e mining possible NoV in ec-
ion. Fo he Subjec 1 NoV in ec ions we e no de ec ed in he
s ool samples collec ed a 4 and 5 mon hs, al hough he
se ocon e sion happened a he six mon hs o age. I is known
ha NoV shedding can be highly a iable, an a e age o 8e60
days [38] and he e o e he in ec ions may be eadily missed i
he samples a e no collec ed weekly. Howe e , GII.2 NoV
in ec ion o he Subjec 2 was de ec ed in a s ool specimen
collec ed p io o blood sample aken a he age o 24 mon hs,
when he i s se ocon e sion was de ec ed. The abo e esul
shows ha al hough a child may be in ec ed wi h a di e se
NoV GII geno ype i se ocon e s o he GII.4 VLPs, as we
ha e p e iously shown [10,11]. NoV-speci ic c oss- eac i e
an ibody epi opes a e p esen in he N- e minal egion o he
Fig. 1. The in eg i y and mo phology o NoV GII.4 VLPs as cha ac e ized by (A) wes e n blo and (B) he elec on mic og aph examined by FEI Tecnai F12
elec on mic oscope (Philips 487 Elec on Op ics, Holland). The p o ein weigh ma ke is shown on he le lane and he pu i ied NoV VLP on he igh lane, using
a human con alescen se um agains NoV GII.4 o de ec ion.
Fig. 2. No o i us GII.4-speci ic se um IgG esponses du ing he i s eigh yea s o li e in Subjec 1. Se um specimens o he child we e collec ed be ween Ma ch,
2000eMa ch, 2008. Se a we e analyzed a 1:100 dilu ion (OD 490 nm) and se ially dilu ed o de e mine he endpoin i e (OD 0.1) o each sample.
266 V. Blaze ic e al. / Mic obes and In ec ion 18 (2016) 263e269
NoV capsid VP1 [39] he e o e, i is possible o de ec se o-
con e sion using VLPs he e ologous o he in ec ing geno ype.
The i e , a idi y and blocking abili y o ma e nal an i-
bodies seemed o in luence suscep ibili y o an in an o NoV
in ec ion. The Subjec 1 had a low le el and low a idi y o
ma e nally acqui ed an ibodies and had he i s NoV in ec ion
ea ly (<6 mon hs o age), while he Subjec 2 wi h high
ma e nal an ibody le el o high a idi y was p o ec ed mo e
han 1.5 yea s. In e es ingly, he Subjec 2 was b eas ed o
only h ee mon hs in con as o he Subjec 1 who was
b eas ed o 25 mon hs indica ing no p o ec i e ole o
b eas eeding agains NoV in ec ion. Bo h child en de eloped
high i e unc ional an ibodies only a he age o wo yea s o
la e , p obably because o he imma u i y o he immune
sys em in an in an [22]. A idi y indexes o sequen ial se um
samples in he p esen s udy con i m he ea lie indings ha
he a idi y is low in p ima y in ec ions and inc eases o e
ime, possibly as a esul o se ial he e o ypic in ec ions [7,23].
Young child en a e able o gene a e homologous p o ec i e
blocking an ibody i e (BT
90
>100) [10,19], which is asso-
cia ed wi h g ea e p o ec ion om NoV in ec ion and illness
[30], bu a e lacking he e ologous blocking esponse and
he e o e a e p one o new in ec ions by ano he NoV s ain
[10]. We ha e ecen ly epo ed ha p e-exis ing blocking
an ibody i e s in acu e se a we e low in <2-yea -old child en
and only child en abo e 12 mon hs o age gene a ed p o ec i e
c oss-blocking an ibodies ollowing he in ec ion, wi h he
endpoin i e >50,000 and BT
90
>100 [19]. In his s udy, a
he ages o ou and six yea s o he Subjec 2 he endpoin IgG
i e o 51,200 and BT
90
100 did no p o ec om new NoV
Table 1
NoV GII.4-1999 speci ic an ibody esponses in Subjec 1. Shown a e GII.4-
speci ic endpoin i e s, GII.4-speci ic blocking i e s (BT) 50 and 90 and
a idi y indexes, (%).
Age
(mon hs/yea s)
P esumed
in ec ion
GII.4 endpoin
i e
Blocking
an ibodies
A idi y
index (%)
c
BT
50
a
BT
90
b
0 m 3200 <50 <50 43
3 m 400 <50 <50 38
6 m x 3200
d
<50 <50 19
12 m 800 <50 <50 25
17 m 400 <50 <50 33
24 m 800 <50 <50 38
3 y x 6400
d
200 100 95
4 y 1600 50 <50 67
5 y x 25,600
d
100 <50 98
6 y 12,800 50 <50 75
7 y 6400 50 <50 82
8 y 12,800 <50 <50 69
a
BT
50
, 50% blocking i e .
b
BT
90
, 90% blocking i e .
c
The a idi y indexes >50% a e shown in bold.
d
>4- old inc ease in an ibody i e .
Fig. 3. No o i us GII.4-speci ic se um IgG esponses in Subjec 2 om bi h o eigh yea s o age. Longi udinal se um samples we e collec ed s a ing om co d
blood on Ap il 9 h, 2000. Se um specimens we e analyzed a 1:100 dilu ion (OD 490 nm) and se ially dilu ed o de e mine he endpoin i e (OD 0.1) o each
sample.
Table 2
NoV GII.4-1999 speci ic an ibody esponses in Subjec 2. Shown a e GII.4-
speci ic endpoin i e s, GII.4-speci ic blocking i e s 50 and 90 and a idi y
indexes, (%).
Age
(mon hs/yea s)
P esumed
in ec ion
GII.4
endpoin i e
Blocking
an ibodies
A idi y
index (%)
c
BT
50
a
BT
90
b
0 m 51,200 200 100 95
3 m 6400 <50 <50 91
6 m 1600 <50 <50 46
17 m 400 <50 <50 42
24 m x 102,400
d
400 100 99
3 y x 204,800 800 400 99
4 y 51,200 200 100 96
5 y x 204,800
d
1600 800 98
5.5 y 51,200 200 100 100
6 y 25,600 100 100 97
8 y x 102,400
d
200 100 97
a
BT
50
, 50% blocking i e .
b
BT
90
, 90% blocking i e .
c
The a idi y indexes >50% a e shown in bold.
d
>4- old inc ease in an ibody i e .
267V. Blaze ic e al. / Mic obes and In ec ion 18 (2016) 263e269

in ec ion (Table 2), con i ming he p e iously de e mined
p o ec i e endpoin i e >50,000 and BT
90
>100 [10].
Ea ly adul expe imen al in ec ion s udies ha e shown
ha NoV immuni y ollowing in ec ion las s up o six
mon hs [21] bu less han wo yea s [20]. Mo e ecen adul
NoV challenge s udies showed du a ion o NoV blocking
an ibodies up o six mon hs [28]. Ou esul s in na u ally
in ec ed child en suppo hese indings. We ound
maximum du a ion o p o ec ion up o 3 yea s a olde age,
which con adic s calcula ions by a ma hema ical model
ha es ima ed ou o eigh yea s'du a ion o p o ec i e
immune esponse o NoV [40]. I is possible ha some in-
ec ions wi h GI o non-GII.4 NoV we e missed in his
s udy as only GII.4-1999 VLPs we e used in he analyses,
bu wha we de ec ed is he minimum numbe o in ec ions.
We ha e ea lie s udied homologous and c oss- eac i e
an ibody esponses in GII.4-2010 New O leans in ec ed
child en [10,19] and ound ha 100% o 6e18 mon h old
child en se ocon e ed o GII.4-2010 NO and GII.4-2012
Sydney [19] and 67% o GII.4-1999, bu only 17% o
mo e dis an GII.12 NoV [10]. Howe e , as he mos com-
mon s ains ci cula ing in Finnish child en a he ime o he
sample collec ion in he p esen s udy we e closely ela ed
o GII.4-1999 (e.g. GII.4-US95/96, GII.4-2001, Fa m-
ing on-2002 and 2006) [14,41], hese in ec ions we e likely
de ec ed by he se ocon e sion o GII.4-1999 VLPs.
P e ious s udies in la ge coho s [24,36] as well as ou
own wo k [11] ha e sugges ed ha a child may encoun e
1e3 NoV in ec ions wi hin he i s yea o li e. The esul s
in he e con i m hese indings and also show ha a leas
h ee in ec ions a e encoun e ed by a child by he age o
i e yea s. Vaccina ion o in an s agains NoV would
add ess he mos common cause o hospi aliza ions due o
gas oen e i is in child en in coun ies whe e uni e sal
o a i us accina ion is implemen ed [42].Ou esul s
sugges ha accine should be deli e ed he ea lies o he
one-yea -old since be o e ha age he immune sys em is
imma u e. Women o a childbea ing age could be consid-
e ed as a accine a ge popula ion o p o ec young in an s.
E en hough his s udy is based on wo subjec s only, he
esul s in his s udy add o he unde s anding o immuno-
genici y and du a ion o p o ec ion in in an s and young
child en.
Con lic o in e es
None o he au ho s ha e con lic o in e es .
Acknowledgmen s
The pe sonnel o he Vaccine Resea ch Cen e , especially
Sanna Ka 
en and Ee a Jokela, and Vi ology Depa men a he
Uni e si y o Tampe e a e acknowledged o echnical assis-
ance. The DIPP s udy pe sonnel a e hanked o he acquisi-
ion o he clinical samples. Financial suppo o DIPP s udy
by Ju enile Diabe es Resea ch Founda ion and Sig id Juselius
Founda ion is acknowledged.
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