pha maceu ics
A icle
Ro a i us VP6 as an Adju an o Bi alen No o i us
Vaccine P oduced in Nico iana ben hamiana
Ma ia Malm 1, And éDiessne 2, Ki si Tamminen 1, Ma kus Liebsche 2, Timo Vesika i 1and
Vesna Blaze ic 1,*
1Vaccine Resea ch Cen e , Facul y o Medicine and Heal h Technology, Tampe e Uni e si y, Bioka u 10,
FI-33520 Tampe e, Finland; [email p o ec ed] (M.M.); [email p o ec ed] (K.T.);
[email p o ec ed] (T.V.)
2Icon Gene ics GmbH, Weinbe gweg 22, 06120 Halle, Ge many; [email p o ec ed] (A.D.);
[email p o ec ed] (M.L.)
*Co espondence: [email p o ec ed]; Tel.: +358-50-421-1054
Recei ed: 21 Ma ch 2019; Accep ed: 5 May 2019; Published: 11 May 2019


Abs ac :
Ro a i uses (RVs) and no o i uses (NoVs) a e majo causes o childhood acu e gas oen e i is.
Du ing de elopmen o a combina ion accine based on NoV i us-like pa icles (VLP) and RV VP6
p oduced in baculo i us exp ession sys em in insec cells, a dual ole o VP6 as a accine an igen
and an adju an o NoV-speci ic immune esponses was disco e ed. He e he VP6 adju an e ec
on bi alen GI.4 and GII.4-2006a NoV VLPs p oduced in Nico iana ben hamiana was in es iga ed.
BALB/c mice we e immunized in ade mally wi h subop imal (0.3
µ
g) dose o each NoV VLP alone
o combined wi h 10
µ
g o VP6, o equal doses o NoV VLPs and VP6 (1
µ
g/an igen). NoV-speci ic
se um IgG an ibodies and hei blocking ac i i y we e analyzed using accine-homologous and
he e ologous NoV VLPs. Immuniza ion wi h 0.3
µ
g NoV VLPs alone was insu icien o induce
NoV-speci ic immune esponses, bu wi h co-adminis a ion o 10
µ
g o VP6, an ibodies agains
accine-de i ed and he e ologous NoV geno ypes we e gene a ed. Fu he mo e, co esponding
adju an e ec o VP6 was obse ed wi h 1
µ
g dose. E icien up ake and p esen a ion o VP6 by
dend i ic cells was demons a ed
in i o
. These esul s show ha adju an e ec o VP6 on bi alen
NoV VLP accine is independen o he cell sou ce used o accine p oduc ion.
Keywo ds:
o a i us; VP6; adju an ; bi alen accine; no o i us; VLP; plan -p oduc ion; blocking
an ibodies
1. In oduc ion
No o i us (NoV) in ec ions a e he mos common cause o acu e gas oen e i is wo ldwide
ac oss all age g oups, causing es ima ed 685 million cases o acu e gas oen e i is (AGE) each yea
and being esponsible o 50,000 yea ly dea hs o child en unde i e yea s o age [
1
,
2
]. The e is
no accine a ailable o NoV, bu p omising candida es a e being de eloped and es ed in clinical
and p eclinical phases [
3
,
4
]. Howe e , in coun ies whe e o a i us (RV) accina ion has no been
in oduced, RV in ec ions a e s ill he mos common cause o AGE- ela ed mo bidi y and mo ali y o
young child en [
5
]. O al RV accines ha e demons a ed good e icacy in high income coun ies, while
in low-income se ings, whe e RV disease is he mos se e e, he e ec i eness o accines is lowe [
6
].
NoV majo capsid p o ein, VP1, spon aneously sel -assembles o o m VLPs ha a e success ully
used as i us pa icle su oga es o accine de elopmen [
3
,
7
–
9
]. Un il ecen ly, he e has been no
app op ia e me hod o NoV p opaga ion and despi e ecen ad ances e.g., wi h en e oid NoV cul u e
sys em [
10
], manu ac u ing scale cul i a ion is s ill lacking. To da e, he e a e a leas 30 human NoV
geno ypes iden i ied, based upon i al capsid (VP1) and RNA-dependen , RNA polyme ase p o ein
Pha maceu ics 2019,11, 229; doi:10.3390/pha maceu ics11050229 www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2019,11, 229 2 o 14
sequences. Mos NoVs in ec ing humans belong o genog oup I (GI, 9 geno ypes) and genog oup II
(GII, 19 geno ypes) [
3
], and he lack o c oss- eac i i y be ween GI and GII NoVs [
8
,
11
,
12
] sugges s ha
inclusion o a leas one VLP om GI and one om GII is necessa y o c oss-p o ec i e NoV accine.
Indeed, he mos ad anced NoV accine in clinical ials [
9
] is a bi alen accine consis ing o NoV
GI.1 and GII.4 VLPs and aluminum hyd oxide [Al(OH)3] as an adju an .
We ha e de eloped a i alen combina ion accine candida e child en as a p ima y a ge g oup
agains NoV and RV AGE, consis ing o wo NoV i us-like pa icles (VLPs), GII.4-1999 and GI.3 and
oligome ic RV VP6 [
7
,
8
]. RV VP6, ~45 kDa in size, is he mos abundan and highly immunogenic
p o ein ha o ms he in e media e laye o he i ion. Fu he mo e, RV VP6 displays a high deg ee o
conse a ion among g oup A RVs ha cause >90% o human RV in ec ions [
13
]. VP6 o ms di e en
oligome ic nanos uc u es such as nano ubes and nanosphe es
in i o
, depending on condi ions
such as pH and ionic s eng h [
14
]. VP6 induced immune esponse has been shown o p o ec om
homologous and he e ologous RV in ec ion in animal models [
15
–
18
]. Fu he mo e, posi i e co ela ion
o se um IgA a ge ed o RV VP6 ollowing bo h RV in ec ion and accina ion has been obse ed
in humans [
19
–
21
]. These cha ac e is ics make VP6 an ideal non-li e RV accine candida e, bu
addi ionally, i has been demons a ed ha RV VP6 has also an adju an e ec on co-deli e ed an igens
such as NoV VLPs [
7
,
22
–
26
]. We ha e p e iously shown ha only when VP6 was co-adminis a ed
in amuscula ly (IM) wi h subop imal dose o ei he GI.3 o GII.4 NoV VLPs in mice, NoV-speci ic
esponse was elici ed [
22
,
23
]. This inding is o a high impo ance as an adju an - ee accine is
p e e ed o pedia ic popula ion.
In he p esen s udy, we e alua ed RV VP6 adju an e ec on a bi alen NoV GI.4 and GII.4-2006a
VLP accine a he han mono alen VLPs s udied be o e [
22
,
23
]. Also, in con as o ou p e ious
s udies using baculo i us (BV)-insec cell p oduced NoV VLPs and RV VP6 all accine an igens used
he e we e p oduced using plan Nico iana (N.)ben hamiana exp ession sys em. Fu he mo e,
in i o
assays using mouse p ima y bone-ma ow-de i ed dend i ic cells (BMDCs) we e pe o med o es
plan -de i ed VP6 in e ac ion wi h an igen-p esen ing cells (APC).
2. Ma e ials and Me hods
2.1. Recombinan P o eins
Plan -de i ed GI.4 and GII.4-2006a NoV VLPs and RV VP6 we e exp essed in N. ben hamiana
plan s using magnICON
®
ec o based on a obacco mosaic i us (TMV) RNA eplicon sys em,
pu i ied and cha ac e ized by ICON Gene ics GmbH (Halle, Ge many) [
27
–
30
]. B ie ly, N. ben hamiana
plan s we e acuum-in il a ed (80–100 mba o 3–4 min) wi h dilu ed Ag obac e ium ume aciens
cul u es wi h TMV-based assembled magnICON
®
ec o s ca ying codon-op imized VP1 (GI.4 o
GII.4-2006a) o VP6 DNA cloned o exp ession and plan ma e ial was ha es ed 6–14 days pos
in il a ion. Biomass was homogenized and cla i ied by cen i uga ion (20 min 15,000
×
g) and
il a ion (Millipo e
®
glass ibe il e AP25). No o i us VLP we e sedimen ed and pu i ied by PEG
p ecipi a ion and il a ion. Ro a i us VP6 high molecula weigh s uc u es we e pu i ied using
a combina ion o ca ion- and anion-exchange ch oma og aphy. Pu i ied ma e ial was o mula ed
in phospha e-bu e ed saline (PBS) (10 mM NaH
2
PO
4
, 137 mM NaCl), pH 7.3. VLP o ma ion
was con i med by size exclusion-high-pe o mance liquid ch oma og aphy (SE-HPLC) wi h ligh
sca e ing analysis. SE-HPLC analysis was pe o med on an Agilen 1200 HPLC sys em (Agilen
Technologies, Waldb onn, Ge many) coupled o a mul iangle ligh sca e ing de ec o (MALS-de ec o )
wi h a quasi-elas ic ligh sca e ing dynamic ligh sca e ing module (QELS-DLS module) and a
e ac ome e (all Wya Technologies Eu ope, De nbach, Ge many). P o ein concen a ion was
measu ed by bicinchoninic acid assay and p o ein pu i y was de e mined wi h educed capilla y gel
elec opho esis pe o med on an Agilen 2100 bioanalyze using a 230 P o ein Ki and 2100 Expe
So wa e (Agilen Technologies, Waldb onn, Ge many). Endo oxin le el was quan i a ed by endpoin
ch omogenic Limulus Amebocy e Lysa e (LAL) es (QCL-1000, Lonza, Walke s ille, MD, USA) and
Pha maceu ics 2019,11, 229 3 o 14
esidual hos cell DNA con amina ion by Quan -iT dsDNA High Sensi i i y Ki (The mo Fishe
Scien i ic, Wal ham, MA, USA). High-o de s uc u es and mo phology o he p o eins we e imaged
by Zeiss EM900 T ansmission Elec on Mic oscope (TEM) (Ca l Zeiss Mic oscopy, Jena, Ge many).
Samples we e collec ed on coope g ids and con as ed wi h 2% u anyl ace a e. Mic og aphs we e
aken wi h a Va iospeed slow scan came a (SM-1k-120, T öndle, Ge many) using he iTEM so wa e
om Olympus SIS (Müns e , Ge many). P o eins we e s o ed a +4
◦
C un il use and dilu ed in
PBS pH 7.3 (Lonza BioWhi ake , Walke s ille, MD, USA, Ca . BE17-516F) and mixed a desi able
concen a ions o immuniza ions. Mock p epa a ion o N. ben hamiana (magnICON
®
) se ed as a
con ol an igen o immunological assays. O he NoV p o ein an igens used o analy ical me hods;
GI.1, GI.3, GII.4-1999, GII.4-2010 NO, GII.4-2012 Sydney, and GII.12 VLPs, we e p oduced in S 9-cells
u ilizing baculo i us-insec cell exp ession sys em, pu i ied and cha ac e ized as desc ibed ea lie by
ou g oup [7,31,32].
2.2. Immuniza ion o Mice
The g oups o i e emale BALB/c OlaHsd mice (En igo, Ho s , The Ne he lands) 7 weeks o age,
we e used o expe imen al and con ol immuniza ions acco ding o Table 1. Vaccine o mula ions we e
adminis a ed in ade mally (ID) a he base o he ail, do sal side o he mouse. Mice we e immunized
wice a day 0 and day 21 and e mina ed a day 35, acco ding o ou s anda d p ocedu e [
7
,
8
,
22
]. Two
di e en doses (0.3
µ
g o 1
µ
g) o a bi alen combina ion o GI.4 and GII.4-2006a NoV VLPs we e
es ed alone (G I and III, Table 1), o mixed wi h RV VP6. The adju an e ec o RV VP6 was i s
e alua ed by combining 10
µ
g VP6 wi h subop imal doses o NoV VLPs (0.3
µ
g, G II) and hen by
adminis a ing equal amoun s (1
µ
g) o each an igen as a mix u e (G IV). Con ol mice (G V) ecei ed
50 µL o phospha e-bu e ed saline (PBS) ca ie only.
Animals we e anes he ized wi h se o lu ane (Bax e Heal hca e L d., Dee ield, IL, USA, Ca .
FDG9117) inhala ion p io o immuniza ion and in ape i oneally wi h a o mula ion o mede omidine
(Do bene
®
e , Labo a o ios SYVA S.A., Leon, Spain, Ca . 067632) and ke amine (Ke aminol
®
e ,
In e e In e na ional B.V., Boxmee , The Ne he land, Ca . 511485) o eu hanasia. Blood samples we e
collec ed a he ime o e mina ion [
33
]. All o he expe imen al p ocedu es ca ied ou we e in acco dance
wi h he egula ions and guidelines o he Finnish Na ional Expe imen Boa d (pe mission numbe
ESAVI/10800/04.10.07/2016) and mice wel a e was moni o ed h oughou he expe imen on a daily basis.
Table 1.
Expe imen al and con ol g oups o immunized mice. Mice we e immunized in ade mally
(ID) a day 0 and 21 wi h he indica ed dose a a olume o 50 µL/injec ion and e mina ed a day 35.
G oup Dose GI.4 +GII.4 Dose VP6 Mice/G oup
I 0.3 µg+0.3 µg - 5
II 0.3 µg+0.3 µg 10 µg 5
III 1 µg+1µg - 5
IV 1 µg+1µg 1 µg 5
V (Con ol) -1- 5
1phospha e bu e ed saline (PBS), ca ie only.
2.3. Se um IgG, IgG1, and IgG2a ELISA
Vaccine-induced NoV GI.4-, GII.4-2006a-, and RV VP6-speci ic an ibodies we e de e mined in
e mina ion blood se um samples acco ding o he p e iously published p ocedu es by enzyme-linked
immunoso ben assay (ELISA) [
7
,
8
]. Type-speci ic an ibodies we e analyzed on Co ning high binding
96-well hal -a ea mic opla es (Co ning Inc., Co ning, NY, USA, Ca . 3690) coa ed wi h 2
µ
g/mL o he
plan -p oduced p o ein an igens. Two- old dilu ed indi idual mouse se a s a ing a 1:200 dilu ion
we e pla ed on blocked (5% milk in PBS) pla es 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, Sain Louis, MO, USA, Ca . A4416),
IgG1 (In i ogen, Ca lsbad, CA, USA, Ca . A10551) o IgG2a (In i ogen, Ca . A10685), ollowed by
Pha maceu ics 2019,11, 229 4 o 14
a eac ion wi h SIGMAFAST o-phenylenediamine dihyd ochlo ide (OPD) subs a e (Sigma-Ald ich,
Sain Louis, MO, USA, Ca . P9187). Op ical densi y a 490 nm (OD490) was measu ed wi h Vic o 2 1420
mic opla e eade (Wallac, Pe kin Elme , Wal ham, MA, USA). The wells o con ol mouse se a we e
used o de e mine he cu -o alue (mean OD490 + h ee s anda d de ia ions (SD)). Specimens wi h a
ne OD490 abo e he se cu -o alue and >0.1 we e conside ed posi i e. The end-poin an ibody i e s
o se a we e de ined as he highes sample dilu ion wi h an OD490 abo e he se cu -o alue. The
se um IgG mock esponses o N. ben hamiana we e es ed acco dingly on pla es coa ed wi h 2
µ
g/mL
wi h mock p epa a ion N. ben hamiana (magnICON
®
). To measu e c oss- eac i e NoV-speci ic IgG
an ibodies in he e mina ion se a, g oup-wise pooled se a we e 1:200 dilu ed and analyzed on pla es
coa ed wi h 1.0
µ
g/mL o baculo i us-p oduced GI.1, GI.3, GII.4-1999, GII.4-2010 New O leans (NO),
GII.4-2012 Sydney (SYD), o GII.12 VLPs ollowing he p ocedu e desc ibed abo e.
2.4. Blocking Assays
The p esence o se um IgG an ibodies ha block binding o NoV VLPs o he HBGA ca bohyd a es
we e de e mined in ELISA-based blocking assay acco ding o p e iously published p o ocols [
23
].
G oup-wise pooled mouse se a we e examined o capabili y o block VLP binding on HBGAs p esen
in pig gas ic mucin (PGM, ype III, Sigma-Ald ich, Sain Louis, MO, USA, Ca . M1778) [
23
,
34
]. Se um
wo- old dilu ions we e p e-incuba ed wi h 0.1
µ
g/mL GI.4 o GII.4-2006a NoV VLPs in sample bu e
(1% milk in PBS +0.05% ween) p io o pla ing on PGM-coa ed (2
µ
g/mL) and blocked (5% milk
in PBS) mic owell pla es (Co ning Inc., Co ning, NY, USA, Ca . 3690). Following 1 h incuba ion a
+37
◦
C, bound VLPs we e de ec ed wi h abbi polyclonal an i-NoV an ise a (ICON Gene ics, Halle,
Ge many) ollowed by an i- abbi IgG-HRP an ibody (Abcam, Camb idge, UK, Ca . ab97051) and
OPD subs a e. Maximum HBGA binding o VLPs was de e mined in wells wi h VLPs lacking he
se um. The blocking index (%) was calcula ed as ollows: 100%
−
[(OD490 o wells wi h VLP and
se um/OD490 o maximum binding wells)
×
100%]. A 50% blocking i e (BT
50
) was de e mined as he
ecip ocal o he highes se um dilu ion able o block a leas 50% o VLP-HBGA binding.
2.5. VP6 In e naliza ion and In acellula S aining
Up ake o plan -based RV VP6 nano ubes by BMDCs was analyzed by in acellula s aining and
low cy ome y ollowing he me hod p e iously desc ibed o immo alized mouse cell lines [
24
] wi h
sligh modi ica ions. BMDCs we e pla ed o non- ea ed mul idish wells in CM con aining 100
µ
g/mL
o VP6 nano ubes o CM alone ( o un ea ed con ol cells) and incuba ed a 37
◦
C, 5% CO
2
o 20 h.
A e he incuba ion he cells we e ha es ed and cul u e supe na an s collec ed and s o ed a
−
80
◦
C
o cy okine analysis. Cells we e washed (PBS +3% FBS) and blocked o non-an igen-speci ic binding
o immunoglobulins o he Fc
γ
III and Fc
γ
II wi h a an i-mouse CD16/CD32 Fc Block (Clone 2.4G2,
Bec on Dickinson, F anklin Lakes, NJ, USA, Ca . 553142). Cells we e ea ed wi h BD Cy o ix/Cy ope m
Plus ki (Bec on Dickinson, Ca . 555028) acco ding o manu ac u e ’s ins uc ions p io o s aining
RV VP6 in acellula ly wi h abbi polyclonal o a i us g oup A an ibody (Genway Bio ech Inc., San
Diego, CA, USA, Ca . GWB-459FC9) ollowed by luo escein iso hiocyana e (FITC)-conjuga ed goa
an i- abbi Ig (Bec on Dickinson, F anklin Lakes, NJ, USA, Ca . 554020). Cells we e esuspended in
FACS S aining Bu e (Bec on Dickinson, F anklin Lakes, NJ, USA, Ca . 554657) o acquisi ion and
analysis using FACS Can oII low cy ome e and FACSDi a So wa e V 6.1.3. The da a analysis was
pe o med using FlowJo so wa e e sion 10.1.
2.6. Cy okine Analysis
Quan i ies o umo nec osis ac o alpha (TNF-
α
) and in e leukin-6 (IL-6) cy okines in he BMDC
cul u e supe na an s we e de e mined by comme cial ELISA ki s, Mouse TNF-
α
DuoSe (R&D Sys ems,
Minneapolis, MN, USA, Ca . DY410-05) and Mouse IL-6 DuoSe (Ca . DY406-05) acco ding o he
manu ac u e s’ ins uc ions as p e iously desc ibed [
24
]. Vic o
2
1420 Mul ilabel Coun e (Wallac,
Pe kinElme , Wal ham, MA, USA) pla e eade was used o op ical densi y eading (OD) o he pla e.
Pha maceu ics 2019,11, 229 5 o 14
Fo each assay he backg ound signal om blank wells (wells wi hou supe na an ) was sub ac ed
om all o he OD eadings on he pla e. S anda d cu es we e plo ed and used o calcula ing he
cy okine concen a ion o each sample (pg/mL).
2.7. ELISPOT IFN-γAssay
To con i m VP6 an igen up ake and p ocessing by an igen-p esen ing cells (APC), an ELISPOT
IFN-
γ
assay was applied. Fi s , BALB/c mouse bone ma ow-de i ed DCs (BMDC) we e gene a ed
and pulsed wi h pu i ied, plan -de i ed VP6 p o ein acco ding o p e iously published me hod [
35
].
Fo pulsing, hawed and washed BMDCs we e pla ed a 2
×
10
6
cells/mL on non- ea ed cell-cul u e
24-well pla es (Co ning Cos a , Co ning, NY, USA, Ca . CLS3738) in cell medium (CM, RPMI-1640,
Ca . R0883, supplemen ed wi h 100 U/mL penicillin and 100
µ
g/mL s ep omycin, Ca . P0781, 2 mm
L-glu amine, Ca . G7513 and 10% e al bo ine se um (FBS), Ca . F9665, all om Sigma-Ald ich and
50
µ
m 2-me cap oe hanol, Gibco 31350-010). BMDCs we e incuba ed ~22 h a +37
◦
C and 5% CO
2
wi h 100 µg/mL VP6, and con ol BMDCs (unpulsed) we e incuba ed in CM only.
RV VP6-speci ic T cell esponses we e analyzed using p e iously published enzyme-linked
immunospo (ELISPOT) IFN-
γ
assay [
8
,
35
]. 96-well Mul iSc een HTS-IP il e pla es (Millipo e,
Bille ica, MA, USA, Ca . MSIPN 4W50) we e coa ed wi h an i-mouse IFN-
γ
(Mab ech Ab, Nacka
S and, Sweden, Ca . 3321-3), washed and blocked wi h 10% FBS in CM be o e pla ing he an igens and
cells. Splenocy es (0.2
×
10
6
cells/well) o mice immunized wi h 10
µ
g BV-de i ed VP6 in a p e ious
s udy we e used as esponde cells in he assay. The esponde cells we e mixed wi h VP6-pulsed
o con ol BMDCs a h ee di e en a ios (40:1, 20:1 o 10:1) Concana alin A (Con A; Sigma-Ald ich,
Sain Louis, MO, USA) 10
µ
g/mL was used as a posi i e con ol o s imula e IFN-
γ
sec e ion om
splenocy es. Pla es we e incuba ed o ~22 h a +37
◦
C and 5% CO
2
, and he spo s we e de eloped wi h
bio inyla ed an i-mouse IFN-
γ
monoclonal an ibody (Ca . 3321-6), 0.5
µ
g/mL in PBS/0.5% FBS, 2 h a RT,
and alkaline-phospha ase (ALP) conjuga ed s ep a idin (Ca . 3310-10) eac ing wi h BCIP/NBT subs a e
(Ca . 3650-10, all om Mab ech, Nacka S and, Sweden). The spo s we e coun ed by an ImmunoSpo
®
au oma ic CTL analyze (CTL-Eu ope GmbH) and he esul s a e exp essed as mean spo o ming cells
(SFCs) pe 10
6
li e splenocy es o eplica e wells. Fo each IFN-
γ
esponse o he pulsed BMDC he
posi i i y cu -o was calcula ed as a mean SFC/106cells o wells wi h con ol BMDC +3×SD.
2.8. S a is ics
A nonpa ame ic Mann-Whi ney U- es was employed o assess he s a is ical di e ences be ween
obse a ions o wo independen g oups. Da a was analyzed wi h G aphPad P ism e sion 8.0.1. The
s a is ically signi ican di e ence was de ined as p<0.05.
3. Resul s
3.1. P o ein Exp ession and Mo phology
Plan -p oduced NoV GI.4 and GII.4-2006a VLPs and RV VP6 p o ein pu i y we e e i ied as
desc ibed in he Ma e ial and Me hods (da a no shown). The in eg i y and mo phology o p o ein
nanopa icles we e con i med unde TEM (Figu e 1A–C). NoV GI.4 (Figu e 1A) and GII.4-2006a
(Figu e 1B) VP1 capsid p o eins had assembled in o VLPs and mos o he VP6 ime s we e associa ed
in o nano ubes (Figu e 1C,D).
3.2. Immune Responses Induced wi h Subop imal Doses o Bi alen No VLP Alone o Combined wi h RV VP6
in Excess
3.2.1. Se um NoV Geno ype-Speci ic IgG, IgG1 and IgG2a
Immuniza ion o mice using subop imal dose o 0.3
µ
g NoV GI.4 and GII.4-2006a VLPs as a mix u e
(G I, Table 1) did no induce se um IgG o GI.4 (Figu e 2A) no o GII.4-2006a (Figu e 2B). Adding

Pha maceu ics 2019,11, 229 6 o 14
10
µ
g o RV VP6 p o ein in o he mix u e (G II, Table 1) ema kably imp o ed NoV-speci ic IgG
esponses o bo h GI.4 (Figu e 2A) and GII.4-2006a (Figu e 2B), wi h signi ican ly inc eased geome ic
mean i e s (GMTs), 1393 o GI.4 (Figu e 2C) and 1838 o GII.4-2006a (Figu e 2D). NoV-speci ic IgG
was no de ec ed in any o he con ol animal se a (Figu e 2A–D).
Pha maceu ics 2019, 11, x FOR PEER REVIEW 6 o 15
IgG esponses o bo h GI.4 (Figu e 2A) and GII.4-2006a (Figu e 2B), wi h signi ican ly inc eased
geome ic mean i e s (GMTs), 1393 o GI.4 (Figu e 2C) and 1838 o GII.4-2006a (Figu e 2D). NoV-
speci ic IgG was no de ec ed in any o he con ol animal se a (Figu e 2A–D).
Figu e 1. Elec on mic og aphs o he highly pu i ied plan -p oduced no o i us (NoV) (A) GI.4 VLPs,
(B) GII.4-2006a VLPs, (C) ecombinan RV VP6 p o ein and (D) a mix u e o he a o emen ioned
examined by ansmission elec on mic oscope EM900 (Ca l Zeiss Mic oscopy, Ge many) ollowing
nega i e s aining wi h 2% u anyl ace a e. The black ba co esponds o 100 nm.
Figu e 2. Se um no o i us (NoV)-speci ic IgG i a ion. Indi idual e mina ion se um o each mouse
in expe imen al g oups immunized wi h 0.3 µg NoV VLPs alone o mixed wi h 10 µg o VP6, and he
con ol g oup (C l, PBS only) we e wo- old dilu ed o analyzing NoV (A) GI.4- and (B) GII.4-2006a-
speci ic IgG an ibodies. Shown a e mean i a ion cu es o each g oup and s anda d e o s o he
Figu e 1.
Elec on mic og aphs o he highly pu i ied plan -p oduced no o i us (NoV) (
A
) GI.4 VLPs,
(
B
) GII.4-2006a VLPs, (
C
) ecombinan RV VP6 p o ein and (
D
) a mix u e o he a o emen ioned
examined by ansmission elec on mic oscope EM900 (Ca l Zeiss Mic oscopy, Ge many) ollowing
nega i e s aining wi h 2% u anyl ace a e. The black ba co esponds o 100 nm.
Pha maceu ics 2019, 11, x FOR PEER REVIEW 6 o 15
IgG esponses o bo h GI.4 (Figu e 2A) and GII.4-2006a (Figu e 2B), wi h signi ican ly inc eased
geome ic mean i e s (GMTs), 1393 o GI.4 (Figu e 2C) and 1838 o GII.4-2006a (Figu e 2D). NoV-
speci ic IgG was no de ec ed in any o he con ol animal se a (Figu e 2A–D).
Figu e 1. Elec on mic og aphs o he highly pu i ied plan -p oduced no o i us (NoV) (A) GI.4 VLPs,
(B) GII.4-2006a VLPs, (C) ecombinan RV VP6 p o ein and (D) a mix u e o he a o emen ioned
examined by ansmission elec on mic oscope EM900 (Ca l Zeiss Mic oscopy, Ge many) ollowing
nega i e s aining wi h 2% u anyl ace a e. The black ba co esponds o 100 nm.
Figu e 2. Se um no o i us (NoV)-speci ic IgG i a ion. Indi idual e mina ion se um o each mouse
in expe imen al g oups immunized wi h 0.3 µg NoV VLPs alone o mixed wi h 10 µg o VP6, and he
con ol g oup (C l, PBS only) we e wo- old dilu ed o analyzing NoV (A) GI.4- and (B) GII.4-2006a-
speci ic IgG an ibodies. Shown a e mean i a ion cu es o each g oup and s anda d e o s o he
Figu e 2.
Se um no o i us (NoV)-speci ic IgG i a ion. Indi idual e mina ion se um o each
mouse in expe imen al g oups immunized wi h 0.3
µ
g NoV VLPs alone o mixed wi h 10
µ
g o VP6,
and he con ol g oup (C l, PBS only) we e wo- old dilu ed o analyzing NoV (
A
) GI.4- and (
B
)
GII.4-2006a-speci ic IgG an ibodies. Shown a e mean i a ion cu es o each g oup and s anda d
e o s o he mean. Dashed line indica es posi i i y cu -o (OD >0.1). IgG end-poin i e s agains (
C
)
GI.4 and (
D
) GII.4-2006a VLPs we e de e mined as he ecip ocal o he highes se um sample dilu ion
gi ing a posi i e eading. Shown a e geome ic mean i e s wi h 95% con idence in e al o each g oup.
G oups we e compa ed by Mann–Whi ney U- es and p alues we e de e mined.
Pha maceu ics 2019,11, 229 7 o 14
Analysis o GI.4- and GII.4-2006a -speci ic se um IgG1 (Figu e 3A,B) and IgG2a (Figu e 3C,D)
showed ha addi ion o VP6 in he mix u e induced inc ease in bo h IgG1 and IgG2a sub ypes, esul ing
in well-balanced Th1/Th2 p o ile agains bo h NoV geno ypes.
To con i m he success o he immuniza ion, all mice ecei ing he VP6 de eloped s ong IgG
an ibody esponse o he p o ein (da a no shown). No posi i e IgG an ibody esponses agains mock
p epa a ion N. ben hamiana (magnICON
®
) we e obse ed in any o he mice immunized wi h he
plan -p oduced p o eins (da a no shown).
Pha maceu ics 2019, 11, x FOR PEER REVIEW 7 o 15
mean. Dashed line indica es posi i i y cu -o (OD > 0.1). IgG end-poin i e s agains (C) GI.4 and (D)
GII.4-2006a VLPs we e de e mined as he ecip ocal o he highes se um sample dilu ion gi ing a
posi i e eading. Shown a e geome ic mean i e s wi h 95% con idence in e al o each g oup.
G oups we e compa ed by Mann–Whi ney U- es and p alues we e de e mined.
Analysis o GI.4- and GII.4-2006a -speci ic se um IgG1 (Figu e 3A,B) and IgG2a (Figu e 3C,D)
showed ha addi ion o VP6 in he mix u e induced inc ease in bo h IgG1 and IgG2a sub ypes,
esul ing in well-balanced Th1/Th2 p o ile agains bo h NoV geno ypes.
To con i m he success o he immuniza ion, all mice ecei ing he VP6 de eloped s ong IgG
an ibody esponse o he p o ein (da a no shown). No posi i e IgG an ibody esponses agains mock
p epa a ion N. ben hamiana (magnICON
®
) we e obse ed in any o he mice immunized wi h he
plan -p oduced p o eins (da a no shown).
Figu e 3. Se um no o i us (NoV)-speci ic IgG1 and IgG2a sub ypes. Pooled e mina ion se a o
expe imen al g oups immunized wi h 0.3 µg NoV GI.4 and GII.4-2006a VLPs alone o mixed wi h 10
µg VP6, and he con ol g oup (C l, PBS only) we e wo- old dilu ed o analyzing NoV (A,C) GI.4
and (B,D) GII.4-2006a-speci ic IgG1 and IgG2a. Shown a e i a ion cu es wi h s anda d e o o he
mean. Dashed line indica es posi i i y cu -o (OD ≥ 0.1).
3.2.2. Se um NoV-Speci ic C oss-Reac i e IgG An ibodies
The p esence o c oss- eac i e IgG an ibodies in pooled se a o immunized expe imen al and
con ol mice was measu ed agains wo VLPs ep esen ing NoV GI (Figu e 4A) and ou VLPs
ep esen ing GII (Figu e 4B). Following adminis a ion o bi alen subop imal dose (0.3 µg pe Ag)
o NoV VLPs alone, c oss- eac i e IgG an ibodies emained unde de ec ion limi (OD < 0.1). When
combining 10 µg VP6 wi h NoV VLPs, c oss- eac i e IgG le el agains all six es ed VLPs geno ypes
could eadily be de ec ed.
Figu e 3.
Se um no o i us (NoV)-speci ic IgG1 and IgG2a sub ypes. Pooled e mina ion se a o
expe imen al g oups immunized wi h 0.3
µ
g NoV GI.4 and GII.4-2006a VLPs alone o mixed wi h
10
µ
g VP6, and he con ol g oup (C l, PBS only) we e wo- old dilu ed o analyzing NoV (
A
,
C
) GI.4
and (
B
,
D
) GII.4-2006a-speci ic IgG1 and IgG2a. Shown a e i a ion cu es wi h s anda d e o o he
mean. Dashed line indica es posi i i y cu -o (OD ≥0.1).
3.2.2. Se um NoV-Speci ic C oss-Reac i e IgG An ibodies
The p esence o c oss- eac i e IgG an ibodies in pooled se a o immunized expe imen al and
con ol mice was measu ed agains wo VLPs ep esen ing NoV GI (Figu e 4A) and ou VLPs
ep esen ing GII (Figu e 4B). Following adminis a ion o bi alen subop imal dose (0.3
µ
g pe Ag)
o NoV VLPs alone, c oss- eac i e IgG an ibodies emained unde de ec ion limi (OD <0.1). When
combining 10
µ
g VP6 wi h NoV VLPs, c oss- eac i e IgG le el agains all six es ed VLPs geno ypes
could eadily be de ec ed.
Pha maceu ics 2019,11, 229 8 o 14
Pha maceu ics 2019, 11, x FOR PEER REVIEW 8 o 15
Figu e 4. Se um no o i us (NoV)-speci ic c oss- eac i e IgG an ibodies. Pooled e mina ion se a o
expe imen al g oups ecei ing 0.3 µg NoV GI.4 and GII.4-2006a VLPs alone o mixed wi h 10 µg o
RV VP6 we e dilu ed 1:200 and es ed o c oss- eac i e IgG agains wo NoV (A) GI VLPs and (B)
ou GII VLPs. Con ol (C l) mice ecei ed ca ie only (PBS). Mean OD alues wi h he s anda d
e o s a e shown o eplica e analysis.
3.2.3. An ibodies Block NoV VLP Binding o PGM HBGAs
Se um NoV-speci ic an ibodies able o block VLP-HBGA binding we e de e mined o mice se a
using PGM HBGA-based blocking assay (Figu e 5A,B). None o he mice immunized wi h
subop imal VLP dose alone o con ol mice had blocking an ibodies a se um dilu ion 1:50. The
adminis a ion o subop imal dose o NoV VLPs combined wi h 10 µg RV VP6 gene a ed
homologous PGM blocking a le el agains GI.4 (Figu e 5A) and GII.4-2006a (Figu e 5B).
Figu e 5. Blocking o no o i us (NoV) VLP binding o his o-blood g oup an igens (HBGAs). Two-
old dilu ed, g oup-wise pooled se a o mice immunized wi h 0.3 µg NoV GI.4 and GII.4-2006a VLPs
alone, o mixed wi h 10 µg VP6, and he con ol (C l) mice se a we e es ed o po en ial o block
binding o NoV (A) GI.4 o (B) GII.4-2006a VLPs o HBGAs p esen in pig gas ic mucin (PGM).
Shown a e g oup mean blocking index (%) wi h s anda d e o o he mean. Ho izon al dashed line
ep esen s 50% blocking.
3.3. RV VP6 Adju an E ec on Bi alen NoV VLP Immune Responses wi h Equal Doses o An igens Used
To u he in es iga e he VP6 adju an e ec on bi alen NoV VLP esponse, he g oups o mice
we e immunized using equal amoun (1 µg) o each an igen. Mice ecei ing NoV GI.4 and GII.4-
2006a VLP alone (G III, Table 1) gene a ed GI.4-speci ic IgG esponse, which did no inc ease wi h
VP6 co-adminis a ion (G IV, Table 1) (p > 0.05) (Figu e 6A). GII.4-2006a-speci ic an ibodies we e
also induced in mice immunized wi h 1 µg o NoV VLPs only (Figu e 6B) (G III), bu he le el was
qui e low (GMT 1212). When 1 µg o VP6 was included o he accine o mula ion (G IV), GII.4-
2006a-speci ic IgG esponse was signi ican ly (p = 0.0317) imp o ed (GMT 12800) (Figu e 6B).
Cong uen ly wi h ype-speci ic IgG i e s, inc ease o c oss- eac i e GII-speci ic IgG an ibodies
Figu e 4.
Se um no o i us (NoV)-speci ic c oss- eac i e IgG an ibodies. Pooled e mina ion se a o
expe imen al g oups ecei ing 0.3
µ
g NoV GI.4 and GII.4-2006a VLPs alone o mixed wi h 10
µ
g o RV
VP6 we e dilu ed 1:200 and es ed o c oss- eac i e IgG agains wo NoV (
A
) GI VLPs and (
B
) ou GII
VLPs. Con ol (C l) mice ecei ed ca ie only (PBS). Mean OD alues wi h he s anda d e o s a e
shown o eplica e analysis.
3.2.3. An ibodies Block NoV VLP Binding o PGM HBGAs
Se um NoV-speci ic an ibodies able o block VLP-HBGA binding we e de e mined o mice se a
using PGM HBGA-based blocking assay (Figu e 5A,B). None o he mice immunized wi h subop imal
VLP dose alone o con ol mice had blocking an ibodies a se um dilu ion 1:50. The adminis a ion o
subop imal dose o NoV VLPs combined wi h 10
µ
g RV VP6 gene a ed homologous PGM blocking a
le el agains GI.4 (Figu e 5A) and GII.4-2006a (Figu e 5B).
Pha maceu ics 2019, 11, x FOR PEER REVIEW 8 o 15
Figu e 4. Se um no o i us (NoV)-speci ic c oss- eac i e IgG an ibodies. Pooled e mina ion se a o
expe imen al g oups ecei ing 0.3 µg NoV GI.4 and GII.4-2006a VLPs alone o mixed wi h 10 µg o
RV VP6 we e dilu ed 1:200 and es ed o c oss- eac i e IgG agains wo NoV (A) GI VLPs and (B)
ou GII VLPs. Con ol (C l) mice ecei ed ca ie only (PBS). Mean OD alues wi h he s anda d
e o s a e shown o eplica e analysis.
3.2.3. An ibodies Block NoV VLP Binding o PGM HBGAs
Se um NoV-speci ic an ibodies able o block VLP-HBGA binding we e de e mined o mice se a
using PGM HBGA-based blocking assay (Figu e 5A,B). None o he mice immunized wi h
subop imal VLP dose alone o con ol mice had blocking an ibodies a se um dilu ion 1:50. The
adminis a ion o subop imal dose o NoV VLPs combined wi h 10 µg RV VP6 gene a ed
homologous PGM blocking a le el agains GI.4 (Figu e 5A) and GII.4-2006a (Figu e 5B).
Figu e 5. Blocking o no o i us (NoV) VLP binding o his o-blood g oup an igens (HBGAs). Two-
old dilu ed, g oup-wise pooled se a o mice immunized wi h 0.3 µg NoV GI.4 and GII.4-2006a VLPs
alone, o mixed wi h 10 µg VP6, and he con ol (C l) mice se a we e es ed o po en ial o block
binding o NoV (A) GI.4 o (B) GII.4-2006a VLPs o HBGAs p esen in pig gas ic mucin (PGM).
Shown a e g oup mean blocking index (%) wi h s anda d e o o he mean. Ho izon al dashed line
ep esen s 50% blocking.
3.3. RV VP6 Adju an E ec on Bi alen NoV VLP Immune Responses wi h Equal Doses o An igens Used
To u he in es iga e he VP6 adju an e ec on bi alen NoV VLP esponse, he g oups o mice
we e immunized using equal amoun (1 µg) o each an igen. Mice ecei ing NoV GI.4 and GII.4-
2006a VLP alone (G III, Table 1) gene a ed GI.4-speci ic IgG esponse, which did no inc ease wi h
VP6 co-adminis a ion (G IV, Table 1) (p > 0.05) (Figu e 6A). GII.4-2006a-speci ic an ibodies we e
also induced in mice immunized wi h 1 µg o NoV VLPs only (Figu e 6B) (G III), bu he le el was
qui e low (GMT 1212). When 1 µg o VP6 was included o he accine o mula ion (G IV), GII.4-
2006a-speci ic IgG esponse was signi ican ly (p = 0.0317) imp o ed (GMT 12800) (Figu e 6B).
Cong uen ly wi h ype-speci ic IgG i e s, inc ease o c oss- eac i e GII-speci ic IgG an ibodies
Figu e 5.
Blocking o no o i us (NoV) VLP binding o his o-blood g oup an igens (HBGAs). Two- old
dilu ed, g oup-wise pooled se a o mice immunized wi h 0.3
µ
g NoV GI.4 and GII.4-2006a VLPs alone,
o mixed wi h 10
µ
g VP6, and he con ol (C l) mice se a we e es ed o po en ial o block binding
o NoV (
A
) GI.4 o (
B
) GII.4-2006a VLPs o HBGAs p esen in pig gas ic mucin (PGM). Shown a e
g oup mean blocking index (%) wi h s anda d e o o he mean. Ho izon al dashed line ep esen s
50% blocking.
3.3. RV VP6 Adju an E ec on Bi alen NoV VLP Immune Responses wi h Equal Doses o An igens Used
To u he in es iga e he VP6 adju an e ec on bi alen NoV VLP esponse, he g oups o mice
we e immunized using equal amoun (1
µ
g) o each an igen. Mice ecei ing NoV GI.4 and GII.4-2006a
VLP alone (G III, Table 1) gene a ed GI.4-speci ic IgG esponse, which did no inc ease wi h VP6
co-adminis a ion (G IV, Table 1) (p>0.05) (Figu e 6A). GII.4-2006a-speci ic an ibodies we e also
induced in mice immunized wi h 1
µ
g o NoV VLPs only (Figu e 6B) (G III), bu he le el was qui e low
(GMT 1212). When 1 µg o VP6 was included o he accine o mula ion (G IV), GII.4-2006a-speci ic
IgG esponse was signi ican ly (p=0.0317) imp o ed (GMT 12800) (Figu e 6B). Cong uen ly wi h
Pha maceu ics 2019,11, 229 9 o 14
ype-speci ic IgG i e s, inc ease o c oss- eac i e GII-speci ic IgG an ibodies agains GII.4 SYD, GII.4
NO and GII.12 we e obse ed when 1
µ
g o VP6 was co-adminis a ed (G IV), bu GI-c oss- eac i e
IgG agains GI.1 o GI.3 NoV VLPs we e no imp o ed (da a no shown).
G oup-wise pooled se um IgG o bo h expe imen al g oups (III, IV) and he con ol g oup was
analyzed o blocking ac i i y o homologous GI.4 (Figu e 6C) and GII.4-2006a (D) VLP binding o
HBGAs p esen in PGM. The e was sligh inc ease in GI.4 VLP blocking ac i i y in se um o mice
immunized wi h VP6 and NoV VLPs (BT
50
=200), compa ed o mice immunized wi h NoV VLPs
only (BT
50
=100) (Figu e 6C). Blocking o GII.4-2006a VLP binding was low when pooled se a o mice
ecei ing only NoV VLPs we e es ed in PGM HBGA blocking assay (BT
50
=50) (Figu e 6D). The
se a o mice immunized wi h VP6-con aining accine o mula ion showed imp o ed GII.4-2006a VLP
blocking ac i i y (BT50 =200) (Figu e 6D).
Pha maceu ics 2019, 11, x FOR PEER REVIEW 9 o 15
agains GII.4 SYD, GII.4 NO and GII.12 we e obse ed when 1 µg o VP6 was co-adminis a ed (G
IV), bu GI-c oss- eac i e IgG agains GI.1 o GI.3 NoV VLPs we e no imp o ed (da a no shown).
G oup-wise pooled se um IgG o bo h expe imen al g oups (III, IV) and he con ol g oup was
analyzed o blocking ac i i y o homologous GI.4 (Figu e 6C) and GII.4-2006a (D) VLP binding o
HBGAs p esen in PGM. The e was sligh inc ease in GI.4 VLP blocking ac i i y in se um o mice
immunized wi h VP6 and NoV VLPs (BT
50
= 200), compa ed o mice immunized wi h NoV VLPs only
(BT
50
= 100) (Figu e 6C). Blocking o GII.4-2006a VLP binding was low when pooled se a o mice
ecei ing only NoV VLPs we e es ed in PGM HBGA blocking assay (BT
50
= 50) (Figu e 6D). The se a
o mice immunized wi h VP6-con aining accine o mula ion showed imp o ed GII.4-2006a VLP
blocking ac i i y (BT
50
= 200) (Figu e 6D).
Figu e 6. Se um no o i us (NoV)-speci ic an ibody esponses induced by equal doses o NoV VLPs
and o a i us (RV) VP6. G oups o mice immunized ei he wi h 1 µg o NoV GI.4 and GII.4-2006a
VLPs o combined wi h 1 µg VP6 we e indi idually es ed o (A) GI.4- and (B) GII.4-2006a-speci ic
se um IgG wi h wo- old dilu ions s a ing a 1:200. Con ol (C l) mice ecei ed ca ie only. Pooled
se um o bo h g oups was es ed o blocking o VLP binding using pig gas ic mucin (PGM)–based
assay. Shown a e blocking i a ion cu es o (C) GI.4 and (D) GII.4-2006a VLP binding. Ho izon al
dashed line ep esen s 50% blocking.
3.4. Plan -Based RV VP6 Is Taken Up and Ac i a es APC
In e naliza ion o plan -based VP6 by BMDC used as APC was analyzed by in acellula s aining
ollowing ~22 h incuba ion o he cells in he p esence o 100 µg/mL RV VP6 p o ein. App oxima ely
33% o BMDCs had in e nalized VP6 du ing he incuba ion pe iod (Figu e 7A, le panel).
P oin lamma o y cy okine sec e ion by APC was measu ed in cul u e media collec ed a he end o
he incuba ion pe iod. VP6 s imula ed BMDCs o p oduce IL-6, as well as TNF-α sec e ion (Figu e
7B). Fu he mo e, APC p ocessing and p esen a ion o in e nalized VP6 o e ec o T cells we e
analyzed in ELISPOT IFN-γ assay using VP6-speci ic T cells as he esponde cells. S ong IFN-γ
esponse by T cells was obse ed co ela ing wi h inc easing numbe o VP6-pulsed BMDC used o
s imula ion o he esponde cells (Figu e 7C).
Figu e 6.
Se um no o i us (NoV)-speci ic an ibody esponses induced by equal doses o NoV VLPs
and o a i us (RV) VP6. G oups o mice immunized ei he wi h 1
µ
g o NoV GI.4 and GII.4-2006a VLPs
o combined wi h 1
µ
g VP6 we e indi idually es ed o (
A
) GI.4- and (
B
) GII.4-2006a-speci ic se um
IgG wi h wo- old dilu ions s a ing a 1:200. Con ol (C l) mice ecei ed ca ie only. Pooled se um
o bo h g oups was es ed o blocking o VLP binding using pig gas ic mucin (PGM)–based assay.
Shown a e blocking i a ion cu es o (
C
) GI.4 and (
D
) GII.4-2006a VLP binding. Ho izon al dashed
line ep esen s 50% blocking.
3.4. Plan -Based RV VP6 Is Taken Up and Ac i a es APC
In e naliza ion o plan -based VP6 by BMDC used as APC was analyzed by in acellula s aining
ollowing ~22 h incuba ion o he cells in he p esence o 100
µ
g/mL RV VP6 p o ein. App oxima ely 33%
o BMDCs had in e nalized VP6 du ing he incuba ion pe iod (Figu e 7A, le panel). P oin lamma o y
cy okine sec e ion by APC was measu ed in cul u e media collec ed a he end o he incuba ion pe iod.
VP6 s imula ed BMDCs o p oduce IL-6, as well as TNF-
α
sec e ion (Figu e 7B). Fu he mo e, APC
p ocessing and p esen a ion o in e nalized VP6 o e ec o T cells we e analyzed in ELISPOT IFN-
γ
assay using VP6-speci ic T cells as he esponde cells. S ong IFN-
γ
esponse by T cells was obse ed
co ela ing wi h inc easing numbe o VP6-pulsed BMDC used o s imula ion o he esponde cells
(Figu e 7C).