Ro a i us capsid VP6 ubula and sphe ical nanos uc u es ac as
local adju an s when co-deli e ed wi h no o i us VLPs
M. Malm, S. Heinim€
aki, T. Vesika i
and V. Blaze ic
Vaccine Resea ch Cen e , Uni e si y o
Tampe e, Tampe e, Finland
Accep ed o publica ion 7 Ap il 2017
Co espondence: Vesna Blaze ic, Bioka u 10,
33520 Tampe e, Finland.
E-mail: [email p o ec ed]
Summa y
A subuni p o ein accine candida e based on no o i us (NoV) i us-like
pa icles (VLPs) and o a i us (RV) VP6 p o ein agains acu e childhood
gas oen e i is has been p oposed ecen ly. RV VP6 o ms di e en
oligome ic nanos uc u es, including ubes and sphe es when exp essed in
i o, which a e highly immunogenic in di e en animal models. We ha e
shown ecen ly ha ecombinan VP6 nano ubes ha e an adju an e ec on
immunogenici y o NoV VLPs in mice. In his s udy, we in es iga ed i he
adju an e ec is dependen upon a VP6 dose o di e en VP6 s uc u al
assemblies. In addi ion, local and sys emic adju an e ec s as well as
equi emen s o an igen co-deli e y and co-localiza ion we e s udied. The
magni ude and unc ionali y o NoV GII.4-speci ic an ibodies and T cell
esponses we e es ed in mice immunized wi h GII.4 VLPs alone o di e en
combina ions o VLPs and VP6. A VP6 dose-dependen adju an e ec on
GII.4-speci ic an ibody esponses was obse ed. The adju an e ec was
ound o be s ic ly dependen upon co-adminis a ion o NoV GII.4 VLPs
and VP6 a he same ana omic si e and a he same ime. Howe e , he
adju an e ec was no dependen on he ypes o oligome s used, as bo h
nano ubes and nanosphe es exe ed adju an e ec on GII.4-speci ic
an ibody gene a ion and, o he i s ime, T cell immuni y. These indings
elucida e he mechanisms o VP6 adju an e ec in i o and suppo i s use
as an adju an in a combina ion NoV and RV accine.
Keywo ds: adju an , nanosphe es, nano ubes, no o i us VLP, o a i us VP6
In oduc ion
No o i uses (NoV) and o a i uses (RV) a e he mos com-
mon causes, a a e y young age, o childhood i al gas o-
en e i is (GE) wo ldwide [1–3]. To elimina e he high isk o
NoV and RV-caused gas oen e i is (GE), a accina ion soon
a e bi h would be needed. Ne e heless, NoV in ec ions
and disease a e no ye p e en able by accina ion, al hough
NoV i us-like pa icles (VLPs)-based accines a e being
s udied ex ensi ely. NoV VLPs a e non-in ec ious sel -
assembled pa icles composed o VP1 capsid p o ein, highly
esembling in ac i ions bo h s uc u ally and an igenically
[4]. Cu en ly used RV accines a e based on li e a enua ed
i uses [5,6] ha a e associa ed wi h po en ial sa e y issues,
such as a isk o in ussuscep ion [7] and shedding and ans-
mission o he accine s ains and easso men o yield
mo e i ulen o ms [8], suppo ing he need o non-li e
RV subuni accines. Ou g oup has ecen ly de eloped a
combina ion accine candida e agains NoV and RV child-
hood GE consis ing o NoV VLPs and RV VP6 [9,10]. The
combina ion accine induced s ong ype-speci ic and c oss-
eac i e humo al and cellula immuni y agains NoV and
RV in a mouse model [9,11–13].
In gene al, highly pu i ied subuni accines wi h he bes
sa e y p o ile ha e poo immunogenici y, and he magni ude
and quali y o he immune esponses need o be enhanced
by adju an s [14]. Howe e , due o he ulne abili y o
in an s and young child en, i would be highly desi able o
ha e an e icien accine wi hou adding ex e nal adju an s
[15,16]. To his end, we ha e shown ha RV VP6 no only
induced p o ec i e immuni y agains li e RV challenge in
mice [13], bu i also ac ed as a s ong in- i o adju an on
he gene a ion o an ibodies speci ic o NoV [17].
In a iple-laye ed RV pa icle he in e media e laye is
o med by he VP6 p o ein (45 kD), si ua ed be ween he
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331
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ou e mos laye consis ing o VP4 and VP7 p o eins and
he inne co e p o ein VP2, which su ounds he double-
s anded genome o RV [6,18,19]. RV VP6 is he mos
abundan and immunogenic RV p o ein [20,21], which is
highly conse ed among RV s ains [22,23]. VP6 o ms
ime s o ganized in o hexagons and packed in o highe -
o de s uc u al assemblies, e.g. VP6 nano ubes (VP6T)
and nanosphe es (VP6S), when exp essed in i o
[12,24–27] unde di e en condi ions [25,28]. The VP6 is
highly immunogenic [9,12,29], and i has also been used as
a ca ie o deli e y pla o m o he e ologous p o ein an i-
gens and gene ically used epi ope-based accines, wi h
imp o ed esponse o he o eign an igen [30–34]. VP6 is
s able a di e en pH condi ions, and when deli e ed o ally
i was a ge ed o in es inal cells, o e ing a p omising new
deli e y pla o m o anspo pha maceu ical compounds
o gas oin es inal ac [35].
In o de o elucida e he mechanisms o VP6 adju an
ac ion, we ha e shown p e iously ha VP6T a e aken up
e icien ly by mac ophages and dend i ic cells (DC) in i o,
esul ing in ac i a ion and ma u a ion o hese an igen-
p esen ing cells (APC). Also, VP6T we e shown o acili a e
he in e naliza ion o co-deli e ed NoV VLPs o he APC
[36]. We unde ook he p esen s udy o in es iga e
equi emen s o co-localiza ion and co-deli e y o he
VP6 wi h he NoV VLPs in i o and whe he VP6 wo ks
as a local o sys emic adju an . In addi ion, adju an e ec
o VP6T was compa ed o VP6S.
Ma e ials and me hods
Recombinan p o eins
NoV VLPs and RV VP6 oligome ic p o eins we e p oduced
in a baculo i us–insec cell exp ession sys em, as desc ibed
in de ail elsewhe e [9,12,37]. NoV GII.4-1999 VLPs
(GenBank e e ence s ain, Accession numbe AF080551)
and VP6 an igens (Accession no. GQ477131) used o
immuniza ions o animals we e highly pu i ied wi h mul i-
s ep ch oma og aphic p ocedu es o a ious s eps o ul a-
il a ion, as desc ibed p e iously [17,38]. The pu i ied
VP6 was assembled in o nano ubes in phospha e-bu e ed
saline (PBS) a pH 73–75 (Lonza, Ve ie s, Belgium) o
nanosphe es in a 50 mM sodium ace a e bu e wi h
130 mM NaCl, pH 482 [38]. The concen a ion o he p o-
eins was de e mined using Pie ce BCA p o ein assay
(The mo Scien i ic, Wal ham, MA, USA). The pu i y o he
p o eins was e i ied by Quan -i dsDNA B oad-Range
Assay Ki (In i ogen, Ca lsbad, CA, USA; <10 ng
dsDNA/10 lg o p o ein), sodium dodecyl sulpha e poly-
ac ylamide gel elec opho esis (SDS-PAGE), BacPAK Rap-
idTi e Ki [Clon ech Labo a o ies, Moun ain View, CA,
USA; 0 plaque- o ming uni s (p u) li e BV/ml] and limulus
amebocy e lysa e assay (Lonza, <01 endo oxin uni s/100
lg o p o ein), as desc ibed in de ail elsewhe e [9,38]. The
VLPs and oligome ic VP6 nanos uc u es used o immu-
niza ions we e con i med by nega i e-s aining ansmission
elec on mic oscopy (TEM) using an FEI Tecnai F12
(Philips Elec on Op ics, Eindho en, he Ne he lands) a e
nega i e s aining wi h 3% u anyl ace a e pH 46 o p o ein
mo phology and in eg i y (Fig. 1a–c). NoV VLPs used o
enzyme-linked immunoso ben assay (ELISA)-based ana-
ly ical me hods, GII.4-1999, GII.4 New O leans (NO) 2009
(Accession no. GU445325) and GII.4 Sydney (SYD) 2012
(Accession no. AFV08795.1) we e pu i ied using suc ose
g adien ul acen i uga ion, as desc ibed elsewhe e [37].
Syn he ic pep ides and i uses
Fo quan i ica ion o NoV-speci ic T cell esponses by
enzyme-linked immunospo (ELISPOT), an in e e on
(IFN)-gassay GII.4 pep ide pool (Synpep ide Co. L d,
Shanghai, China) con aining 76 syn he ic pep ides [18-
me s, 11 amino acid (aa) o e lap], spanning he en i e
539aa sequence o GII.4-1999 NoV VP1 [39], was used.
VP6-speci ic BALB/c mouse (H-2
d
) CD4
1
T cell epi ope
242
DGATTWYFNPVILRPNNV
259
[11,40], named R6-2,
was syn he ized (P oimmune L d, Ox o d, UK) and used
Fig. 1. S uc u e and in eg i y o he p o eins. Elec on mic oscopy images o baculo i us–insec cell sys em-p oduced no o i us (NoV) GII.4-1999
i us-like pa icles (VLPs) (a), o a i us (RV) VP6 nano ubes (b) and VP6 nanosphe es (c) examined by FEI Tecnai F12 elec on mic oscope (Philips
Elec on Op ics) a e nega i e s aining wi h 3% u anyl ace a e, pH 46. Images obse ed a 323 000 (a,c) o 36800 (b) magni ica ion.
M. Malm e al.
332 V
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o Immunology, Clinical and Expe imen al Immunology, 189: 331–341
in RV VP6-speci ic ELISPOT assays. O albumin (OVA)
323–339 chicken egg albumin pep ide (aa
323
ISQAVHAA-
HAEINEAGR
339
, ca . # ac-isq; In i ogen, San Diego, CA,
USA) se ed as a nega i e con ol.
Fo RV-speci ic ELISPOT assays human RV s ains Wa
(G1P1A[8]) and bo ine RV s ain WC3 (G6P7[5]) we e
p opaga ed in e al hesus monkey kidney (MA104) cells,
as desc ibed p e iously [12], and Ridasc een
V
R
Ro a i us ki
(R-Biopha m AG, Da ms ad , Ge many; ca . C0901) wi h
he in e nal VP6 s anda d was used o de e mine he VP6
amoun (ng/ml) in he cul u es.
Immuniza ion o animals
Female 7–8-week-old BALB/c mice ob ained om En igo
Labo a o ies (Ho s , Limbu g, he Ne he lands) we e
di ided o nine g oups (g oups I–IX) and immunized
in amuscula ly (i.m.) a s udy weeks 0 and 3 wi h di e en
doses o an igens dilu ed in 50 ml PBS, as shown in Table 1.
A minimum o i e mice in each g oup was used. All an i-
gens we e deli e ed in o he igh high muscle o each
mouse excep g oup VI, which ecei ed GII.4 VLPs a he
igh high and VP6T a he con ala e al le limb si e a
he same ime. Fo immunizing g oups II–IV and VII, he
VLPs and VP6 we e mixed p io o adminis a ion. To
de ine he empo al equi emen s o VP6 adju an ac i i y,
mice in g oup V ecei ed 10 mg VP6T i s , ollowed by 03
mg VLP 1 h la e a he same si e. Nai e mice ecei ing ca -
ie only (g oup IX) we e used as nega i e con ols. Ani-
mals we e anaes he ized be o e immuniza ion wi h
se o lu ane inhala e and o eu hanasia wi h a mix u e o
mede omidine (Do bene e , 1 mg/ml; Labo a o ios SYVA
SA, Leon, Spain) and ke amine (Ke aminol e , 50 mg/ml;
In e e In e na ional BV, Boxmee , he Ne he lands).
Blood and spleens we e collec ed a he ime o e mina ion
(a week 5) o he analysis o se ological and cell-media ed
immune esponses, as desc ibed p e iously [41]. Se um
samples we e s o ed a 2208C, and spleen cell suspensions
we e s o ed in liquid ni ogen be o e use in he assays. All
mice used we e nega i e o NoV GII.4-speci ic immuno-
globulin (Ig)G an ibodies p io o he immuniza ions a
week 0 (da a no shown). 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 guidelines o he Finnish Na ional Ani-
mal Expe imen Boa d.
Se um IgG an ibody ELISA
Se a o immunized and con ol mice we e analysed indi-
idually by ELISA o de e mine NoV GII.4-1999, GII.4-
NO, GII.4 SYD and VP6-speci ic IgG, IgG1 and IgG2a
i es, as desc ibed p e iously [9,11]. B ie ly, 96-well pla es
(Co ning Inc. Co ning, NY, USA) we e coa ed wi h 50 ng/
well o NoV VLPs o VP6 in PBS. Duplica es o wo old
se ial dilu ions o se um samples we e incuba ed o 2 h a
oom empe a u e and bound an ibodies we e de ec ed
wi h ho se adish pe oxidase (HRP)-conjuga ed goa an i-
mouse IgG (Sigma-Ald ich, S Louis, MO, USA), IgG1
(In i ogen) o IgG2a (In i ogen). Op ical densi y (OD)
a 490 nm was measu ed by a mic opla e eade Vic o
2
1420 (Pe kin Elme , Wal ham, MA, USA) and a sample was
conside ed posi i e i he OD was abo e he mean OD o
con ol mice 13 s anda d de ia ions (s.d.) and >01. End-
poin i es we e exp essed as he highes se um dilu ion
gi ing a posi i e eading.
Blocking assay (a su oga e neu aliza ion assay)
To examine he abili y o se um an ibodies o block he
binding o NoV VLPs o a pu a i e cellula his o-blood
g oup an igen (HBGA) ecep o , wo di e en sou ces o
HBGAs, pig gas ic mucin (PGM) ype III (Sigma Chemi-
cals, S Louis, MO, USA) and human ype A sali a we e
u ilized [42,43]. The blocking assays we e conduc ed wi h
he p ocedu es desc ibed in de ail elsewhe e [44]. B ie ly,
g oupwise pooled wo old se um dilu ions we e p eincu-
ba ed wi h he 01mg/ml GII.4-1999 VLPs and he mix u es
we e added o 96-mic owell pla es coa ed wi h 25mg/ml
PGM o 1 : 3000 dilu ed human ype A sali a. Maximum
Table 1. Expe imen al and con ol immuniza ion g oups
G oup Dose o GII.4 VLP Dose and o m o VP6 Adminis a ion
I03mg – i.m.
II 03mg1mg VP6T i.m., co-adminis a ion
III 03mg10mg VP6T i.m., co-adminis a ion
IV 03mg30mg VP6T i.m., co-adminis a ion
V03mg10mg VP6T i.m., VP6 i s ollowed by
he VLP a e 1 h
VI 03mg10mg VP6T i.m., le igh VP6, igh igh
VLP, a he same ime
VII 03mg10mg VP6S i.m., co-adminis a ion
VIII 3 mg – i.m.
IX (C l) – – i.m., ca ie only (PBS)
VLP 5 i us-like pa icle; VP6 5 ecombinan VP6; i.m. 5in amuscula ; VP6T 5VP6 nano ubes; VP6S 5VP6 nanosphe es; C l 5con ol;
PBS 5phospha e-bu e ed saline.
Ro a i us VP6 ubula and sphe ical nanos uc u es as local adju an s
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333
VLP binding signal (OD) was de e mined in wells lacking
se um. The bound VLPs we e de ec ed using human an i-
NoV de ec ion se um and an i-human IgG conjuga e
(No ex; The mo Fishe Scien i ic, F emon , CA, USA).
Blocking index was calcula ed as ollows: 100% 2[OD
(wells wi h se um)/OD (wells wi hou se um, maximum
binding)] 3100%. Resul s a e exp essed as he blocking
i e 50 (BT
50
), a se um i e blocking 50% o he VLPs
binding o he HBGAs [45].
No o i us-speci ic ELISPOT IFN-g
To quan i y NoV GII.4-speci ic IFN-g-p oducing T cells o
immunized mice, an ELISPOT assay was used [11]. Spleno-
cy es om ei he indi idual o g oupwise pooled mice we e
pla ed on Mul iSc eenHTS-IP il e pla es (Millipo e, Bille -
ica, MA, USA) coa ed wi h an an i-mouse IFN-gmonoclo-
nal an ibody (Mab ech AB, Nacka S and, Sweden) a 5 mg/
ml and blocked wi h 10% e al bo ine se um (FBS; Sigma-
Ald ich). Cells we e s imula ed wi h GII.4-99 pep ide pool
(2 mg/ml), GII.4-99 VLPs (5 mg/ml) o OVA pep ide (nega-
i e con ol, 4 mg/ml). Cells incuba ed in cul u e medium
(CM) only (RPMI 1640 supplemen ed wi h 10% e al
bo ine se um, 100 U/ml penicillin, 100 mg/ml s ep omycin,
50 mM 2-me cap oe hanol and 2 mM L-glu amine; Sigma-
Ald ich) and cells we e s imula ed wi h 10 mg/ml o T cell
mi ogen concana alin A (ConA; Sigma-Ald ich, ca . C5275)
se ed as a backg ound and iabili y con ol. A e o e nigh
incuba ion (16–20 h) IFN-gwas de ec ed wi h bio inyla ed
an i-mouse IFN-gmonoclonal an ibody (Mab ech AB, ca .
3321–6) and s ep a idin–alkaline phospha ase (ALP) conju-
ga e (Mab ech AB, ca . 3310–10). The spo s we e de eloped
wi h BCIP/NBT (5-b omo-4-chlo o-3-indolyl-phospha e)
subs a e (Mab ech AB, ca . 3650–10) and coun ed by
ImmunoSpo
V
R
au oma ic CTL analyse (CTL-Eu ope
GmbH, Bonn, Ge many). The esul s we e exp essed as
mean spo - o ming cells (SFC)/10
6
splenocy es o duplica e
wells. A posi i e esul was conside ed as an inc ease o wice
o mo e abo e he con ol an igen SFC coun s. Backg ound
coun s ne e exceeded 20 SFC/10
6
cells.
RV VP6-speci ic IFN-gand IL-4 ELISPOT
VP6-speci ic IFN-gand in e leukin (IL)-4 T cell esponses
we e es ed using an ELISPOT assay s imula ing he spleno-
cy es o immunized and con ol mice wi h VP6 p o ein (5
mg/ml), R6-2 pep ide (5 mg/ml), RV Wa, RV WC3 (05mg
VP6/ml) o MA104 mock an igen. IFN-gELISPOT assay
was pe o med simila ly o NoV-speci ic ELISPOT IFN-g,as
desc ibed abo e. Fo quan i ica ion o IL-4 p oduc ion he
Mul iSc eenHTS-IP il e pla es we e coa ed wi h an i-
mouse IL-4 monoclonal an ibody (Mab ech AB, ca . 3311-3)
a 5 mg/ml. G oupwise pooled cells we e pla ed and incu-
ba ed wi h he an igens o 40–45 h. Bio inyla ed an i-mouse
IL-4 an ibody (Mab ech AB, ca . 3311-6) ollowed by incu-
ba ion wi h s ep a idin–ALP and BCIP/NBT subs a e we e
used o de eloping he spo s. The spo s we e coun ed and
he esul s we e exp essed as desc ibed abo e.
S a is ics
The s a is ical di e ences be ween independen g oups
we e assessed by Mann–Whi ney U- es o se um IgG end-
poin i es and ELISPOT assay esul s. Wilcoxon’s signed-
ank es o compa ing wo ela ed samples was used o
analysing he di e ence be ween IgG1 and IgG2a end-poin
i es. S a is ical analyses we e pe o med using IBM SPSS
s a is ics (SPSS, Chicago, IL, USA) e sion 23. P<005 was
conside ed s a is ically signi ican .
Resul s
VP6 adju an e ec on NoV-speci ic se um an ibody
esponses is dose-dependen
In o de o de e mine he mechanism o adju an e ec o
RV VP6 on NoV-speci ic an ibody esponses, mice we e
immunized wi h a subop imal dose (03lg) o GII.4-1999
VLP alone o co-adminis e ed wi h di e en doses o
VP6T. Immuniza ion wi h 03mg GII.4 VLP did no elici
a signi ican se um IgG esponse, whe eas co-
adminis a ion o 03mg GII.4 VLP wi h 10 mg VP6T
esul ed in obus NoV-speci ic se um IgG le els (Fig. 2a),
con i ming ou p e ious obse a ion [17]. The GII.4-spe-
ci ic se um IgG i es we e simila (P50853) o he i es
ob ained wi h a 3 lg dose o GII.4 VLP alone used as a pos-
i i e con ol (Fig. 2a, g oup VIII). A 1 lg dose o VP6T co-
adminis e ed wi h 03lg GII.4 VLP induced lowe an i-
body esponses han a 10 lg dose o VP6T (Fig. 2a, g oups
II and III, P<001), while a 30 lg dose induced simila
esponses (Fig. 2a, g oups III and IV, P50425), indica ing
ha he VP6 adju an e ec was dose-dependen , bu pla-
eaued a e maximum e ec .
VP6 adju an e ec on NoV-speci ic se um an ibody
esponses is local and equi es co-deli e y
The spa ial and empo al equi emen s o VP6 adju an
ac i i y we e nex e alua ed. A equi emen o co-
adminis a ion o 03mg GII.4 VLP 110 mg VP6T was
es ed by deli e ing an igens a he same ime as a mix u e
(Table 1, g oup III), deli e ing he VLPs 1 h ollowing he
VP6T a he same si e (Table 1, g oup V) o deli e ing an i-
gens a he same ime a he con ala e al si es (Table 1,
g oup VI). Co-adminis a ion as a mix u e induced signi i-
can ly highe se um NoV GII.4-speci ic IgG han injec ing
VP6T and GII.4 VLPs sepa a ely in o he same si e wi h 1 h
di e ence (Fig. 2a, g oups III and V, P50005). Rema k-
ably, no VP6 adju an e ec on GII.4-speci ic se um IgG
was seen when he an igens we e spa ially dissocia ed
(Fig. 2a, g oups III and VI, P<0005). Hence, he adju an
M. Malm e al.
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o Immunology, Clinical and Expe imen al Immunology, 189: 331–341
e ec o VP6 is appa en ly exe ed locally a he si e o
adminis a ion and no sys emically.
VP6 adju an e ec on NoV-speci ic se um an ibody
esponses is no dependen upon he ype o
oligome ic s uc u es
We u he de e mined i he e is a di e ence in VP6 adju-
an e ec induced wi h VP6T o VP6S, as bo h ubula as
well as sphe ical s uc u es we e shown p e iously o be
highly immunogenic in mice [12]. When 10 lg VP6S we e
co-deli e ed wi h he VLPs ins ead o he VP6T,
compa able IgG i es we e obse ed (P50211) (Fig. 2a),
indica ing ha bo h oligome ic con o ma ional s uc u es
o VP6 ha e compa able adju an abili y. All nega i e con-
ol mice ecei ing PBS only we e nega i e o NoV GII.4-
speci ic an ibodies (da a no shown).
VP6 p omo es unbiased Th1/Th2- ype esponses
To de e mine i he VP6 has an adju an e ec on NoV
GII.4-speci ic T helpe ype 1 (Th1)- ype and/o Th2- ype
esponses, expe imen al g oups whe e he VP6 adju an
e ec was seen (Fig. 2a, g oups III, IV and VII, espec i ely)
Fig. 2. GII.4 geno ype-speci ic immunoglobulin (Ig)G an ibody i es. IgG (a), IgG1 (b) and IgG2a (c) end-poin i es we e de e mined om
indi idual, se ially dilu ed se a o immunized mice in enzyme-linked immunoso ben assay (ELISA). Ba s ep esen log
10
geome ic mean i es
wi h 95% con idence in e als. Fo nega i e mice se a, an a bi a y i e o 1 : 100 (hal he s a ing se um dilu ion, 1 : 200) was assigned.
*Signi ican ly di e en (P<005) an ibody i es compa ed o g oup III (a) o compa ed o g oup I (b,c).
Ro a i us VP6 ubula and sphe ical nanos uc u es as local adju an s
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o Immunology, Clinical and Expe imen al Immunology, 189: 331–341
335

we e es ed o GII.4-speci ic IgG1 and IgG2a an ibody
sub ypes. Bo h GII.4-speci ic IgG1 (Fig. 2b) and IgG2a
an ibodies (Fig. 2c) we e de ec ed cong uen ly o he IgG
esponses seen in Fig. 2a, wi h signi ican ly highe end-
poin i es induced when VP6 was co-adminis a ed wi h
03mg VLP (P<005 o all). The e we e no signi ican di -
e ences in he sub ype-speci ic se um end-poin i es
obse ed (P50125), indica ing balanced Th1- and
Th2- ype esponses.
VP6 adju an e ec on NoV-speci ic c oss- eac i e
and blocking an ibodies
The VP6 adju an e ec on c oss- eac i e se um IgG i es
agains wo di e en a ian s o NoV GII.4 VLPs, GII.4
NO-2010 and GII.4 SYD-2012 VLPs was also in es iga ed
(Fig. 3). C oss- eac i e IgG an ibodies ollowing immuni-
za ion wi h 03mg GII.4 VLPs we e imp o ed signi ican ly
(P<0005) by co-adminis a ion wi h 10 mg (g oup III) o
30 lg (g oup IV) o VP6T o 10 lg VP6S (g oup VII). The
VP6 adju an e ec on NoV GII.4-speci ic blocking an i-
bodies was in es iga ed u he . Human ype A sali a and
PGM we e used as he HBGA sou ces o NoV GII.4 VLP
binding. The e was no VLP binding blocking (BT
50
525)
by he mouse se a immunized wi h he subop imal 03lg
dose o VLP alone (Table 2). When VLPs we e co-
adminis a ed wi h 10 o 30 lg o VP6T o VP6S, BT
50
o
100–200 we e obse ed (Table 2). As expec ed, due o he
lack o GII.4-speci ic se um an ibodies, he 10 mgo VP6T
Fig. 3. No o i us (NoV) c oss- eac i e immunoglobulin (Ig)G an ibodies. Indi idual, se ially dilu ed se a o immunized mice we e analysed o
c oss- eac i e an i-GII.4 New O leans (NO) and an i-GII.4 Sydney (SYD) IgG end-poin i es. An a bi a y end-poin i e o 50 (hal he
s a ing dilu ion, 1 : 100) was assigned o nega i e mice se a. Ba s ep esen log
10
-exp essed geome ic mean i es o each g oup wi h 95%
con idence in e als. *Signi ican ly di e en (P<005) an ibody i es compa ed o g oup III.
Table 2. Blocking an ibody i es in immunized mouse se a
G oup Immuniza ion
Sali a HBGA PGM HBGA
BT
50
BT
50
I03mg VLP 25
a
n. .
II 03mg VLP 11mg VP6T 50 50
III 03mg VLP 110 mg VP6T 200 200
IV 03mg VLP 130 mg VP6T 200 100
V03mg VLP 110 mg VP6T (1 h) 50 50
VI 03mg VLP 110 mg VP6T
(con ala e al si es)
25 n. .
VII 03mg VLP 110 mg VP6S 100 100
VIII 3 mg VLP 400 n. .
IX (C l) Ca ie only (PBS) 25 25
a
BT
50
o 25 was assigned o samples lacking blocking a a se um dilu ion 1:50.
HBGA 5his o-blood g oup an igen; PGM 5pig gas ic mucin; BT
50
5 i es wi h 50% blocking o no o i us (NoV) GII.4-1999 VLP binding;
VLP 5 i us-like pa icle; n. . 5no es ed; VP6T 5VP6 nano ubes; VP6S 5VP6 nanosphe es; C l 5con ol; PBS 5phospha e-bu e ed saline.
M. Malm e al.
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o Immunology, Clinical and Expe imen al Immunology, 189: 331–341
and 03lg VLP adminis e ed a he con ala e al si e
(g oup VI) as well as he con ol g oup (g oup IX) did no
induce blocking an ibodies (Table 2).
VP6 adju an e ec on NoV-speci ic T cell esponses
NoV GII.4-speci ic T cell esponses we e analysed by s imu-
la ing he splenocy es o immunized mice wi h GII.4-1999-
speci ic pep ide pool and GII.4-1999 VLPs and measu ing
IFN-gp oduc ion in he ELISPOT assay (Fig. 4). Immuni-
za ion o mice wi h a 03mg dose o GII.4 VLPs alone did
no induce de ec able IFN-gp oduc ion, bu when co-
adminis a ed wi h 10 mg o VP6T o VP6S a conside able
NoV GII.4-speci ic IFN-gsec e ion was obse ed (Fig. 4).
The e was no signi ican di e ence in he o e all IFN-g
p oduc ion by he T cells o mice immunized wi h VP6T o
VP6S (P>0.05). Mice immunized wi h he 10 mg o VP6T
and 03lg VLP a he con ala e al si es (g oup VI, Table
1) we e nega i e o all an igens es ed in he ELISPOT
IFN-g(da a no shown), con i ming he local adju an
e ec o VP6 as seen o he NoV GII.4-speci ic an ibody
esponses (Fig. 2a).
RV VP6-speci ic se um an ibody esponses
Each g oup o mice immunized wi h VP6 (g oups II–VII,
Table 1) a di e en doses o di e en oligome ic s uc u es
de eloped RV VP6-speci ic se um IgG an ibodies, indica -
ing success ul immuniza ion (Fig. 5a). In e es ingly, e y
high VP6-speci ic IgG end-poin i es (GMT 60887) we e
induced in mice immunized wi h GII.4 VLPs and VP6T a
he con ala e al si es (g oup VI, Table 1), uling ou he
possibili y ha a lack o VP6 adju an e ec obse ed in
Fig. 4. No o i us (NoV) GII.4-speci ic in e e on (IFN)-g
p oduc ion by T cells. NoV GII.4-1999-speci ic pep ide pool, GII.4-
1999 i us-like pa icles (VLPs) and o albumin (OVA) pep ide
(nega i e con ol) we e used o s imula ing IFN-gp oduc ion o
he mice immunized wi h 03mg VLP alone o co-adminis a ed
wi h 10 mg VP6 nano ubes (VP6T) o nanosphe es (VP6S).
Nega i e con ol mice (C l) we e immunized wi h phospha e-
bu e ed saline (PBS) only. Resul s a e exp essed as he mean spo -
o ming cells (SFC) pe 10
6
splenocy es o he duplica e wells wi h
s anda d e o s o he mean. The expe imen s we e epea ed wo o
mo e imes wi h simila esul s.
Ro a i us VP6 ubula and sphe ical nanos uc u es as local adju an s
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o Immunology, Clinical and Expe imen al Immunology, 189: 331–341
337
his g oup migh esul om an unsuccess ul immuniza-
ion wi h VP6.
RV VP6-speci ic T cell esponses
ELISPOT IFN-g(a hallma k o a Th1- ype esponse) and
IL-4 (a hallma k o a Th2- ype esponse) we e used o ana-
lyse RV VP6-speci ic T cell esponses in splenocy es o
immunized mice o VP6 de i ed R6-2 pep ide epi ope, RV
cell cul u e an igens (Wa and WC3) o VP6 p o ein. IFN-
gp oduc ion o all an igens bu o a mock an igen was
de ec ed in mice immunized by co-adminis a ion o
03lg NoV GII.4 VLPs and 10 lg VP6T o VP6S (Fig. 5b).
Immuniza ion wi h VP6T induced somewha highe IFN-g
p oduc ion han VP6S, bu he di e ence was no s a is i-
cally signi ican (P50073). When he same cells we e
es ed o VP6-speci ic IL-4 p oduc ion, a obus IL-4
esponse (up o 1016 SFC/10
6
cells) was obse ed in he
g oup ha ecei ed 03mg NoV VLPs co-adminis e ed wi h
10 mg o VP6T (Fig. 5c). IL-4 p oduc ion was also induced
in he g oup ha ecei ed 10 mg o VP6S (Fig. 5c) ins ead
o VP6T; howe e , he esponse was signi ican ly highe in
he g oup ha ecei ed VP6T (P50.008). No RV VP6-
speci ic IFN-go IL-4 was sec e ed by he splenocy es o
mice immunized wi h 03mg GII.4 VLP alone (da a no
shown) o he con ol mice (Fig. 5b,c).
Discussion
We ha e demons a ed ecen ly an in- i o adju an e ec
o VP6T on he immunogenici y o NoV VLPs [17]. In
addi ion, in- i o s udies ha e shown ha VP6 induces
APC ac i a ion and ma u a ion [36]. In he p esen s udy,
he mechanism o VP6 adju an ac ion was in es iga ed
u he o explo e he e ec o co-localiza ion o he wo
an igens, NoV VLPs and VP6, ia co-deli e y as a mix u e
o as sepa a e injec ions a he same o con ala e al injec-
ion si es.
An adju an is de ined as a compound ha enhances he
immune esponse o accine an igens. The adju an s may
be ega ded as depo s o ehicles o /and immunomodula-
o y agen s. A depo e ec acili a es and imp o es deli e y
o an igens o APCs. We he e o e in es iga ed i he VP6
unc ions as a ca ie o deli e y ehicle ha would necessi-
a e he VLP and VP6 co-deli e y as a mix u e, ins ead o
sepa a e injec ions. The esul s demons a ed clea ly ha
he adju an e ec o VP6 is s ic ly dependen upon co-
localiza ion o he VP6 wi h NoV GII.4 VLPs. To accom-
plish he op imal VP6 adju an e ec on NoV VLP immu-
nogenici y i was essen ial o co-adminis a e he VP6 wi h
he NoV VLPs, as bo h empo al and spa ial dissocia ion o
VP6 and NoV VLPs impai ed o comple ely abolished he
adju an e ec . A 1-h empo al di e ence in he adminis-
a ion o VP6T and NoV VLPs impai ed he adju an
e ec d ama ically, u he suppo ing he ole o VP6 as a
deli e y ehicle. I has been published ha pa icula e an i-
gens a el e y quickly, in e ms o minu es, om he si e
o injec ion o he local lymphoid issue [46]. Al hough
his s udy does no ex end o con i m his, he esul s sug-
ges ha RV VP6 and VLPs may o m agg ega es when co-
o mula ed, VP6 unc ioning as a ca ie . Indeed, ou
ecen ly published esul s showed ha NoV VLP up ake
in o he APCs was inc eased when mixed wi h he VP6
[36].
In addi ion o ac ing ini ially as he deli e y ehicle o
NoV VLPs he VP6 adju an e ec is p obably en o ced
u he by APC ac i a ion. A e being deli e ed, VP6 may
induce local cy okine and chemokine p oduc ion and ac i-
a ion and ma u a ion o APC a he si e, as shown by ou
ea lie in- i o s udies [36]. P oin lamma o y cy okines,
such as umou nec osis ac o (TNF)-a, IL-6, IL-1 and
g anulocy e mac ophage colony-s imula ing ac o (GM-
CSF), enhance he adap i e immune esponse in se e al
ways, including ec ui men o APC a he si e o injec ion,
s imula ing hei ma u a ion and mig a ion o lymph
nodes (LN) [47,48]. The e o e, he VP6 deli e y p obably
leads o an inc eased numbe o NoV VLP-loaded APCs in
he LN d aining he injec ion si e able o ac i a e an igen-
speci ic T cells. Th ee ypes o DCs, con en ional and
monocy e-de i ed in s eady s a e skele al muscles a e
a ge ed by i.m. immuniza ion, which can encoun e he
an igen and mig a e o LN [49]. Fu he mo e, VP6 ac i-
a ion o T cells in lymphoid issues induces cy okine
p oduc ion by hese cells, such as he obse ed IFN-g,a
Th1- ype cy okine and IL-4, a Th2- ype cy okine. Pa a-
c ine sec e ion o Th1 and Th2 cy okines by he VP6-
speci ic T cells is o signi icance, as hese cy okines d i e
p oli e a ion and di e en ia ion o co-deli e ed an igen;
in his case NoV VLPs p imed nai e B and T lympho-
cy es in o memo y cells [50,51]. The localized na u e o
he VP6 adju an ici y is suppo ed by he obse a ion
Fig. 5. Ro a i us (RV) VP6-speci ic an ibodies and T cell esponses.
VP6-speci ic immune esponses we e de ec ed ollowing
immuniza ion wi h 03mg no o i us (NoV) GII.4 i us-like pa icles
(VLPs) oge he wi h VP6 nano ubes (VP6T) o nanosphe es
(VP6S). Con ol (C l) mice ecei ed phospha e-bu e ed saline
(PBS) only. (a) Se um an i-VP6 IgG o indi idual mice we e es ed
in enzyme-linked immunoso ben assay (ELISA) and he mean
i a ion cu es o each expe imen al g oup a e shown. Ba s
ep esen log
10
geome ic mean i es wi h 95% con idence in e als.
Fo nega i e mice se a, an a bi a y i e o 1 : 100 (hal he s a ing
se um dilu ion, 1 : 200) was assigned. VP6-speci ic IFN-g(b) and
in e leukin (IL)-4 (c) p oduc ion by T cells was es ed s imula ing
he cells wi h VP6-speci ic R6-2 pep ide, RV Wa and WC3 cell
cul u e an igens, mock an igen o ecombinan VP6 p o ein ( VP6).
Resul s a e exp essed as he mean spo - o ming cells (SFC)/10
6
splenocy es o he duplica e wells wi h s anda d e o s o he mean.
The expe imen s we e epea ed wo o mo e imes wi h simila
esul s.
3
M. Malm e al.
338 V
C2017 The Au ho s. Clinical & Expe imen al Immunology published by John Wiley & Sons L d on behal o B i ish Socie y
o Immunology, Clinical and Expe imen al Immunology, 189: 331–341
ha VP6 adminis a ion a he con ala e al si e did no
con ibu e o he NoV-speci ic immuni y, e en hough
high VP6-speci ic immune esponses we e obse ed.
Mo eo e , se um collec ed a 3 and 24 h pos -VP6T
immuniza ion was nega i e o p oin lamma o y cy o-
kines TNF-aand IL-6 by ELISA (da a no shown),
a guing agains a sys emic adju an e ec .
The esul s o he p esen s udy show ha VP6 p o-
mo es bo h Th1- and Th2- ype esponses o NoV wi hou
skewing he o e all immune esponse in any pa icula
di ec ion. The adju an s a e mainly es ic ed o enhance
ei he ype o he esponses, such as aluminium sal s, which
p omo e mainly Th2- ype esponses o he co-
adminis e ed accine an igens [52]. The pa icle size and
shape a e shown o be cen al in he an igen in e naliza ion
and anspo o LN and immune cell ac i a ion
[48,53–55]. Addi ionally, he pa icle size and shape may
play a ole in uning he adap i e immuni y, as od-shaped
pa icles ha e been epo ed o induce Th2-biased
esponses compa ed o sphe ical pa icles [56]. Howe e ,
al hough we obse ed mo e IL-4 p oduc ion by he VP6T-
speci ic T cells compa ed o VP6S, bo h hese nanos uc-
u es exe ed a simila adju an e ec on NoV GII.4-spe-
ci ic an ibody esponses.
Ou esul s show, o he i s ime, he VP6 adju an
e ec on NoV-speci ic T cell immuni y. I has been pub-
lished ha VP6 can po en ia e he se um an ibody esponse
agains RV in a mouse model by an in e molecula help
mechanism [57]. In he p esen s udy, as he VP6 is no
conjuga ed o linked o he VLPs, bys ande T cell help is
p obably an impo an mechanism. These cells may d i e
p oli e a ion and di e en ia ion o NoV-an igen p imed B
and T lymphocy es in o memo y cells and an ibody-
sec e ing plasma cells ei he by cell- o-cell con ac
(CD40-CD40L in e ac ion) [58] o soluble cy okine p o-
duc ion [59].
Al oge he , his s udy shows clea ly ha co-
adminis a ion o he VP6 wi h NoV VLPs is essen ial o
he op imal adju an e ec on NoV-speci ic immune
esponses and ha he VP6 ac s as a deli e y ehicle as well
as immunomodula o . Sa e y is a majo conce n when i
comes o adju an app o al o human use, especially o
heal hy in an s [16]. The esul s o his s udy suppo he
use o VP6 no only o p o ide p o ec ion agains RV in
he p oposed NoV VLP–RV VP6 combina ion accine
[9,10], bu also as an adju an o po en ia e he NoV VLP
immunogenici y.
Acknowledgemen s
We g a e ully acknowledge Ki si Tamminen and 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. No ex e nal
unding was ecei ed o his s udy.
Au ho con ibu ions
M. M. conduc ed immuniza ions, sample acquisi ion and
p ocessing and labo a o y analysis, including da a acquisi-
ion, analysis and in e p e a ion (ELISA, blocking assays),
w i ing he manusc ip and inal app o al o he e sion o
be submi ed. S. H. conduc ed immuniza ions, sample
acquisi ion, p ocessing and labo a o y analysis, da a acqui-
si ion and analysis (p o ein and i us p oduc ion, ELI-
SPOT assays). Final app o al o he e sion o be
submi ed. T. V. is he head o he Vaccine Resea ch Cen e
and conduc ed c i ical e iew o he pape and ga e inal
app o al o he e sion o be submi ed. V. B. is he head o
he labo a o y and co esponding au ho , and conduc ed
he 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 he
pape o impo an in ellec ual con en and inal app o al
o he e sion o be submi ed.
Disclosu e
All he au ho s decla e no disclosu es.
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