BRIEF REPORT
P o ec ion agains li e o a i us challenge in mice induced
by pa en e al and mucosal deli e y o VP6 subuni o a i us
accine
Su i Lappalainen
1
•Ana Ru h Pas o
2
•Ma ia Malm
1
•Vanessa Lo
´pez-Gue e o
3
•
Fe nando Esqui el-Guada ama
3
•Lau a A. Paloma es
2
•Timo Vesika i
1
•
Vesna Blaze ic
1
Recei ed: 24 Feb ua y 2015 / Accep ed: 20 May 2015 / Published online: 29 May 2015
ÓThe Au ho (s) 2015. This a icle is published wi h open access a Sp inge link.com
Abs ac Li e o al o a i us (RV) accines a e pa o
ou ine childhood immuniza ion bu a e associa ed wi h
ad e se e ec s, pa icula ly in ussuscep ion. We ha e de-
eloped a non-li e combined RV – no o i us (NoV) ac-
cine candida e consis ing o human RV inne -capsid VP6
p o ein and NoV i us-like pa icles. To de e mine he
e ec o deli e y ou e on induc ion o VP6-speci ic p o-
ec i e immuni y, BALB/c mice we e adminis e ed a ac-
cine con aining RV VP6 in amuscula ly, in anasally o a
combina ion o bo h, and challenged wi h mu ine RV. A
leas 65 % p o ec ion agains RV shedding was obse ed
ega dless o deli e y ou e. The le els o pos -challenge
se um VP6-speci ic IgA i e s co ela ed wi h p o ec ion.
Keywo ds Ro a i us VP6 IgA In anasal 
In amuscula P o ec ion
Ro a i us (RV) causes se e e gas oen e i is in in an s and
child en unde 5 yea s o age wi h high mo ali y and
mo bidi y a es [1]. Cu en ly, wo li e o al RV accines,
he mono alen Ro a ix
Ò
(GlaxoSmi hKline) and he pen-
a alen Ro a eq
Ò
(Me ck), a e licensed and used ex en-
si ely [2,3]. Howe e , hese o al accines a e less
e icacious in de eloping coun ies [4,5] and a e associa ed
wi h sa e y conce ns such as a isk o in ussuscep ion [6].
Non-li e subuni RV accines a e he e o e conside ed as
al e na i es o RV immuniza ion.
Co ela es o p o ec ion agains RV in ec ion a e no
ully unde s ood. Type-speci ic neu alizing an ibodies
agains he ex e nal p o eins VP4 and VP7 ha e a ole in
p o ec i e immuni y a e na u al RV in ec ion [7,8], bu
hei ole in accine-induced p o ec i e immuni y agains
se e e RV gas oen e i is has no been shown. Al hough
se um an i-RV an ibody IgA i e s as a co ela e o p o-
ec ion ha e been dispu ed [9], he bes su oga e ma ke o
RV accine-induced p o ec ion appea s o be a high le el o
se um RV IgA an ibody a ge ed o he inne capsid p o ein
VP6 [10,11], which de e mines i al g oup (A-H) and
subg oup (SGI, II, I?II, non-I/II o g oup A) speci ici y
[12] and is highly conse ed [13], immunogenic [14,15]
and he mos abundan RV p o ein [12]. VP6 does no in-
duce classical neu alizing an ibodies, bu i induces
he e o ypic c oss- eac i e p o ec ion in mice [16–18].
No o i us (NoV) is ano he leading cause o acu e gas-
oen e i is in child en, wi h genog oups GI and GII being
esponsible o he majo i y o NoV cases [19]. Fo p o ec-
ion agains childhood gas oen e i is, we ha e in oduced a
concep o accina ion agains RV and NoV wi h a com-
bined i alen accine consis ing o RV VP6 p o ein and
NoV GI.3 and GII.4 i us-like pa icles (VLPs) [20]. We
ha e p e iously shown ha a candida e combina ion accine
deli e ed in amuscula ly (IM) o mice was highly im-
munogenic [20], and in anasal (IN) immuniza ion p o ec ed
mice agains mu ine RV challenge [21]. Deli e y equi e-
men s o he NoV componen s in he induc ion o p o ec i e
NoV immune esponse we e published ecen ly [22]. In his
wo k, we compa ed IM and IN deli e y and he combina ion
o bo h o induc ion o VP6-speci ic p o ec i e immuni y
agains RV challenge, and we examined humo al immune
esponses o co ela ion wi h p o ec ion.
&Vesna Blaze ic
[email p o ec ed]
1
Vaccine Resea ch Cen e , Uni e si y o Tampe e Medical
School, Bioka u 10, 33520 Tampe e, Finland
2
Ins i u o de Bio ecnologı
´a, Uni e sidad Nacional Au o
´noma
de Me
´xico, Cue na aca, Mo elos, Me
´xico
3
Facul ad de Medicina, Uni e sidad Au o
´noma del Es ado de
Mo elos, Cue na aca, Mo elos, Me
´xico
123
A ch Vi ol (2015) 160:2075–2078
DOI 10.1007/s00705-015-2461-8
Human RV VP6 p o ein (SGII) used o immuniza ion
and as an igen in ELISA was p oduced using a baculo i us
exp ession sys em in S 9 insec cells [23]. The i alen
RV-NoV combina ion accine was p epa ed by mixing he
VP6 ubules and NoV GI.3 and GII.4 VLPs in equal
amoun s [20].
Female 7-week-old BALB/c OlaHsd mice (5
mice/g oup) (Ha lan, Ho s , The Ne he lands) we e im-
munized IM o IN wice (a s udy weeks 0 and 3) wi h he
i alen accine con aining 10 lg o RV VP6 pe im-
muniza ion poin . Mo eo e , sequen ial IM and IN immu-
niza ions (4 mice/g oup) wi h 10 lg o VP6 alone we e
pe o med o de e mine whe he adminis a ion a wo
dis inc si es would enhance p o ec ion. No ex e nal adju-
an s we e used. Naı
¨ e mice ecei ing PBS se ed as
con ols. P e-immune (week 0) and p e-challenge (week 5)
ail blood samples o indi idual mice we e collec ed, p o-
cessed o ob ain se a and dilu ed 1:100 in PBS. A week 6,
mice we e challenged o ally wi h 1 910
4
ocus- o ming
uni s (FFU) (100 imes he dia heal dose DD
50
) o he
mu ine RV s ain EDIM
w
(SG non-I/II, G3P10[16]),
o iginally ob ained om D . Wa d (Gamble Ins i u e o
Medical Resea ch, Cincinna i, OH). Fecal samples we e
collec ed p io o challenge (day 0) and daily o 8 days
(days 1-8) a e he challenge. Mice we e eu hanized a day
8, when whole blood samples we e also collec ed. The
p o ocol o he s udy (pe mission numbe 167-2010) was
app o ed by he Bioe hics Commi ee o he Ins i u o de
Bio ecnologia (Uni e sidad Nacional Au o
´noma de
Me
´xico).
RV VP6-speci ic p e- and pos -challenge an ibody e-
sponses we e de e mined by measu ing le els o an i-VP6
IgG and IgA in indi idual se a a 1:100 and wo- old di-
lu ion se ies by ELISA acco ding o p e iously published
p ocedu es [20,21].
The p esence o RV an igen in ecal samples was de-
e mined using an an igen ELISA [16]. Fecal an igen
shedding was exp essed as he ne OD
405
alue ( he OD o
he p e-challenge ecal sample sub ac ed om he OD o
he pos -challenge samples o he indi idual mouse).
The p e-immune se a o all mice we e nega i e o an i-
VP6 IgG and IgA (da a no shown). Robus sys emic IgG
esponses we e induced by each immuniza ion ou e
(Fig. 1a). Geome ic mean i e s (GMTs) o se um IgG
achie ed by he IM, IN and IM?IN ou es we e equi alen
(p=0.663). IN and IM?IN deli e y elici ed de ec able
IgA an ibodies (p=0.556), while IM immuniza ion did
no (Fig. 1b). No an i-VP6 an ibodies we e de ec ed in se a
o con ol mice p io o he challenge (Fig. 1a and b).
The quan i y o RV an igen shed in ecal samples was
de e mined up o 8 days pos -challenge (Fig. 2a). A sig-
ni ican di e ence in i al shedding was de ec ed be ween
he mice immunized IM, IN and IM?IN and he con ol
mice (p=0.011), whe eas he shedding be ween he im-
munized g oups was no di e en (p=0.514). The o al
an igen shedding o mice immunized IM and IN dec eased
66 % (±12 %) and 65 % (±18 %) compa ed o he con-
ols (Fig. 2a and b). Al hough sequen ial IM?IN immu-
niza ion con e ed a nume ically highe p o ec ion a e
(84 ±5 %) (Fig. 2b), i was no s a is ically di e en om
he g oups immunized IM o IN.
No co ela ion o p e-challenge i e s o IgG
( =-0.455, p=0.127) o IgA ( =-0.198, p= 0.497)
an ibodies wi h p o ec ion a es was de ec ed. A e he RV
challenge, VP6-speci ic se um IgG and IgA an ibody i e s
inc eased in all VP6-immunized mice (Fig. 1a and b), bu
only he le els o he pos -challenge IgA inc eased sig-
ni ican ly compa ed o he p e-challenge le els (p 0.03).
P o ec ion le els co ela ed wi h he le els o se um IgA
a e he challenge ( =0.607, p=0.006). Following he
challenge, con ol mice also de eloped low le els o IgG
Fig. 1 P e- and pos -challenge VP6-speci ic IgG (a) and IgA
(b) an ibodies in se a o indi idual mice immunized IM and IN wi h
he i alen accine con aining VP6 (5 mice/g oup) o sequen ially
IM?IN wi h VP6 (4 mice/g oup). A sample was conside ed ELISA
posi i e i he op ical densi y a 490 nm (OD
490
) was abo e he se
cu o alue (mean OD
490
o con ol mice ?39SD) and C0.1. All
con ol mice we e combined (8 mice/g oup). Endpoin i e s o
indi idual mice, exp essed as log
10
o he ecip ocal o he highes
sample dilu ion gi ing a posi i e eading, as well as geome ic mean
i e s o he g oups (———) a s udy weeks 5 (p e-challenge ail-
blood sample) and 7 (pos -challenge e mina ion se a) a e shown. A
i e o 50 was assigned o all nega i e samples, being a hal o he
s a ing se um dilu ion. The s a is ical di e ences be ween non-
pa ame ic obse a ions o independen g oups we e assessed by
Mann-Whi ney U- es (SPSS Inc, Chicago, IL); pB0.05 was
conside ed o indica e a s a is ically signi ican di e ence
2076 S. Lappalainen e al.
123
(GMT B2.5 log
10
) and IgA (GMT 2 log
10
), bu he i e s
we e signi ican ly lowe han hose o he accina ed mice
(p 0.001).
RV VP6 has been p oposed as a subuni accine can-
dida e agains RV by us [14,20,21,23] and o he s [17]. I
o ms di e en oligome ic s uc u es in i o [24], which
a e highly immunogenic in mice wi hou he need o ex-
e nal adju an s [14,20,21,25]. Due o he epe i i e
mul i alen an igenic s uc u es, hese oligome s a e able o
c oss-link B-cell ecep o s e y e icien ly [26], whe eas
soluble VP6 gene ally equi es an adju an o induc ion o
an immune esponse [17]. Al hough he ole o VP6 in
p o ec i e immuni y is s ill unclea , VP6 may be su icien
o p o ec i e immuni y, as induc ion o p o ec ion agains
RV in ec ion in mice and abbi s has been achie ed wi h
inac i a ed double-laye ed (dl) RV pa icles [27], dl2/6-
VLPs [28] and VP6 p o ein [17,21,25] wi hou he su ace
VP4 and VP7 an igens. Unlike he su ace p o eins, an i-
bodies o he inne capsid VP6 a e non-neu alizing.
Howe e , an i-VP6 IgA, bu no IgG, is able o inhibi RV
eplica ion in acellula ly [18,29].
Human RV-de i ed VP6 p o ein gi en pa en e ally o
mucosally induced simila le els o p o ec ion agains RV
EDIM
w
in ec ion. P o ec ion was e alua ed in an adul
mouse model, which is an in ec ion model bu no a disease
model, by measu ing educ ion in ecal RV an igen shed-
ding a e i al challenge [30]. Immunized mice showed
signi ican educ ion ([65 %) in i us shedding when
compa ed o he con ols. The p o ec ion was incomple e
bu o he o de o magni ude ha is achie ed agains any
RV disease in humans a e li e RV accina ion. These
esul s indica e e icacy o he VP6-based accine in
con e ing p o ec i e immuni y agains li e RV challenge
independen ly o he deli e y ou e. Simila educ ion a es
we e p e iously published o mice immunized subcu a-
neously wi h VP6 ubules [25]. Pa ial p o ec ion was also
achie ed wi h inac i a ed dl RV pa icles [27] and VP6
DNA accines a e IM adminis a ion [31]. P o ec ion
close o 100 % agains shedding o wo mu ine RV s ains
has been elici ed a e IN immuniza ion wi h MBP-VP6
only a e inclusion o an ex e nal adju an [17].
Al hough in es inal IgA was shown o be c i ical o RV
clea ance and p o ec ion in he mouse model [32], se um
RV IgA a ge ed o VP6 has been conside ed he bes
su oga e ma ke o accine-induced p o ec ion in humans
[10,11]. We de ec ed a posi i e co ela ion be ween pos -
challenge VP6-speci ic se um IgA le els and he RV p o-
ec ion a e in mice. Bo h pa en e al and mucosal deli e y
induced simila clea ance o RV, e en hough only he IN
and IM?IN ou es led o de ec able p e-challenge se um
IgA an ibodies. IM immunized mice may ha e had unde-
ec able p e-exis ing se um IgA le el, which expanded
apidly a e i al eplica ion in he gu [33]. Vi al epli-
ca ion possibly led o a signi ican inc ease in se um IgA
i e s in VP6-p imed mice, which co ela ed wi h educ ion
in RV an igen shedding and he e o e p o ec ion. Howe e ,
e idence o a co ela ion o se um IgA wi h p o ec ion has
been con adic o y in animal models [34]. By con as ,
co ela ion o p o ec ion wi h se um IgA has been p e-
sen ed in mice ollowing IN immuniza ion wi h dl2/6-
VLPs and chole a oxin [28].
In conclusion, he human RV VP6 p o ein induced
conside able p o ec ion in mice agains li e he e ologous
RV challenge, independen ly o he immuniza ion ou e.
These esul s highligh he impo ance o non-se o ype-
speci ic an ibody esponses induced using he highly con-
se ed VP6 p o ein in he e o ypic p o ec ion.
Acknowledgmen s We g a e ully acknowledge he echnical as-
sis ance gi en by he labo a o y pe sonnel o he Vaccine Resea ch
Cen e o Uni e si y o Tampe e Medical School and he Animal
Facili y o Ins i u o de Bio ecnologı
´a (Uni e sidad Nacional
Au o
´noma de Me
´xico).
Fig. 2 P o ec ion agains RV shedding in immunized mice. Vi al
shedding cu es (OD
405
e sus day pos -challenge) o each animal
we e plo ed and he educ ion in i al load was calcula ed by
compa ing he mean a ea unde he shedding cu e o he immunized
mice o he mean a ea unde he cu e o he con ols. a. Vi al
shedding cu es o expe imen al g oups. Each poin ep esen s he
daily a e age o an igen shed pe g oup wi h s anda d e o o he
mean. As e isks (*) indica e a signi ican di e ence (pB0.05; Mann-
Whi ney U- es ) in daily shedding be ween he immunized and
con ol mice. b. Reduc ions in i us shedding o VP6-immunized
mice ollowing challenge. Mean pe cen educ ions o he expe imen-
al g oups wi h s anda d e o o he means a e shown. A [50 %
educ ion in i us shedding was conside ed signi ican p o ec ion
om i us challenge, as epo ed p e iously
P o ec ion o mice by VP6 subuni o a i us accine 2077
123
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