SHORT COMMUNICATION
A Coxsackie i us B accine p o ec s agains i us-induced diabe es
in an expe imen al mouse model o ype 1 diabe es
Vi ginia M. S one
1,2
&Minna M. Hankaniemi
2
&Emma S edin
1
&
Ami babak Sioo y-Khojine
2
&Sami Oika inen
2
&Heikki Hyö y
2,3
&Olli H. Lai inen
2
&
Vesa P. Hy önen
2,3
&Malin Flods öm-Tullbe g
1,2
Recei ed: 30 July 2017 /Accep ed: 4 Oc obe 2017 /Published online: 18 No embe 2017
#The Au ho (s) 2017. This a icle is an open access publica ion
Abs ac
Aims/hypo hesis Epidemiological s udies sugges a ole o
Coxsackie i us B (CVB) se o ypes in he pa hogenesis o
ype 1 diabe es, bu hei ac ual con ibu ion emains elu-
si e. In he p esen s udy, we ha e p oduced a CVB1
accine o es whe he accina ion agains CVBs can
p e en i us-induced diabe es in an expe imen al model.
Me hods NOD and SOCS1- g mice we e accina ed h ee
imes wi h ei he a o malin- ixed non-adju an ed CVB1 ac-
cine o a bu e con ol. Se um was collec ed o measu emen
o neu alising an ibodies using a i us neu alisa ion assay.
Vaccina ed and bu e - ea ed mice we e in ec ed wi h CVB1.
Vi aemia and i al eplica ion in he panc eas we e measu ed
using s anda d plaque assay and PCR. The de elopmen o
diabe es was moni o ed by blood glucose measu emen s.
His ological analysis and immunos aining o i al capsid
p o ein 1 (VP1), insulin and glucagon in o malin- ixed
pa a in embedded panc eas was pe o med.
Resul s The CVB1 accine induced s ong neu alising
an ibody esponses and p o ec ed agains i aemia and
he dissemina ion o i us o he panc eas in bo h NOD
mice (n=8)andSOCS1- g mice (n= 7). Con e sely,
100% o he bu e - ea ed NOD and SOCS1- g mice we e
i aemic on day 3 pos in ec ion. Fu he mo e, hal (3/6)
o he bu e - ea ed SOCS1- g mice de eloped diabe es
upon in ec ion wi h CVB1, wi h a loss o he insulin-
posi i e be a cells and damage o he exoc ine panc eas.
In con as , all (7/7) accina ed SOCS1- g mice we e
p o ec ed om i us-induced diabe es and showed no
signs o be a cell loss o panc eas des uc ion (p<0.05).
Conclusions/In e p e a ion CVB1 accine can e icien ly
p o ec agains bo h CVB1 in ec ion and CVB1-induced
diabe es. This p eclinical p oo o concep s udy p o ides
a base o u he s udies aimed a de eloping a accine o
use in elucida ing he ole o en e o i uses in human ype 1
diabe es.
Keywo ds An ibody .Coxsackie i us .En e o i us .Mouse
model .NOD mice .Type 1 diabe es .Vaccine
Abb e ia ions
CVB Coxsackie i us B
GMK G een monkey kidney
PFU Plaque o ming uni s
p.i. Pos in ec ion
VP1 Vi al capsid p o ein 1
In oduc ion
Type 1 diabe es is inc easing globally, howe e , he mecha-
nisms ha ini ia e he onse o disease emain unknown.
En i onmen al ac o s ha ha e been implica ed include en-
e o i us in ec ions, in pa icula Coxsackie i us B (CVB) se-
o ypes [1,2]. CVBs a e common RNA i uses encompassing
Elec onic supplemen a y ma e ial The online e sion o his a icle
(h ps://doi.o g/10.1007/s00125-017-4492-z) con ains pee - e iewed bu
unedi ed supplemen a y ma e ial, which is a ailable o au ho ised use s.
*Malin Flods öm-Tullbe g
malin. lods om- ullbe [email protected]
1
The Cen e o In ec ious Medicine, Depa men o Medicine,
Ka olinska Ins i u e , Ka olinska Uni e si y Hospi al Huddinge, F59,
SE-141 86 S ockholm, Sweden
2
Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e,
Tampe e, Finland
3
Fimlab Labo a o ies, Pi kanmaa Hospi al Dis ic , Tampe e, Finland
Diabe ologia (2018) 61:476–481
h ps://doi.o g/10.1007/s00125-017-4492-z
six se o ypes; CVB1–6. Usually in ec ions a e mild o asymp-
oma ic, howe e , some lead o se e e, po en ially a al dis-
eases including asep ic meningi is and myoca di is.
Epidemiological s udies exis documen ing associa ions be-
ween CVBs and ype 1 diabe es, including he p esence o
i us in he panc eas o indi iduals ecen ly diagnosed wi h
ype 1 diabe es and inc eased occu ence o in ec ion p io o
isle an ibody appea ance and onse o diabe es [1–3].
Fu he mo e, CVBs can induce hype glycaemia in animal
models [4,5]. Toge he , hese indings suppo he hypo hesis
ha CVBs con ibu e o he pa hogenesis o ype 1 diabe es.
Despi e he a o emen ioned ci cums an ial e idence, i is
possible ha he associa ion be ween CVBs and ype 1 diabe-
es is no causal and esul s om ye uniden i ied con ounding
ac o s. Vaccine s udies in p ospec i e bi h coho s o gene -
ically suscep ible child en may help elucida e he ole o
CVBs in ype 1 diabe es, howe e , no comme cially a ailable
CVB accines cu en ly exis [2]. We p e iously demons a -
ed ha an inac i a ed, non-adju an ed CVB1 accine was
well ole a ed in mice, was highly e icacious agains CVB1
in ec ion and did no accele a e diabe es in NOD mice [6,7].
Whe he his accine can p e en CVB1-induced diabe es is
unknown.
SOCS1- g mice exp ess he supp esso o cy okine
signalling-1 in be a cells, esul ing in hei inabili y o espond
o in e e ons, hus lea ing be a cells suscep ible o CVB in-
ec ion, des uc ion and subsequen ly i us-induced diabe es
[4]. Due o i s obus ness and quick de elopmen o i us-
induced diabe es, he SOCS1- g mouse model was used o
assess he abili y o a CVB1 accine o p e en CVB1 induced
diabe es, p o iding an impo an biological p oo o concep
s udy examining CVB accine e icacy in he con ex o ype
1diabe es.
Me hods
Animal husband y and moni o ing o animal heal h NOD
mice and SOCS1- g mice on a NOD backg ound (bo h om
in-house b eeding) we e housed in speci ic pa hogen- ee con-
di ions a Ka olinska Ins i u e , S ockholm, Sweden. E hics
app o al was g an ed o all expe imen s by he local e hics
commi ee and we e conduc ed in acco dance wi h he NIH
P inciples o Labo a o y Animal Ca e and na ional laws in
Sweden. Ex ended heal h moni o ing o mice was pe o med.
Addi ional in o ma ion is p o ided in he ESM Me hods:
Animals, oge he wi h de ails o p ime s used o SOCS1- g
mouse geno yping (ESM Table 1).
Vi us and accine p oduc ion A CVB1 ield isola e (CVB1-
V200; [3]), was p opaga ed in Ve o cells (ECACC no.
84113001; mycoplasma nega i e), pu i ied and hen o malin
inac i a ed o 3 days a 37°C o p oduce CVB1 accine. See
ESM Me hods: Vaccine p oduc ion and Hankaniemi e al [7]
o mo e de ails. Mice we e in ec ed wi h CVB1-10796 (p op-
aga ed in HeLa cells, o iginally ob ained om R Glas,
Ka olinska Ins i u e , S ockholm, Sweden, mycoplasma
nega i e).
Vaccina ions Male and emale age-ma ched NOD and
SOCS1- g mice (4–7 weeks old) we e andomly assigned o
g oups and accina ed on days 0, 14 and 28 wi h non-
adju an ed accine con aining 1.8 μg p o ein (n=8and
n= 7, espec i ely) o mock- accina ed wi h accine bu e
(M199-0.1% Tween80 ( ol./ ol.), 150 μl, n= 6 o NOD and
o SOCS1- g mice) by in e scapula injec ion [6,7]. Se um
was collec ed be o e accina ions and in ec ion (day 35) by
ail bleeding (expe imen al imeline displayed in Fig. 1a). The
s udy was no blinded o he expe imen e .
In ec ions Mice we e challenged wi h 10
6
plaque o ming
uni s (PFU) CVB1-10796 (dilu ed in se um- ee RPMI o a
inal olume o 200 μl, adminis e ed by in ape i oneal injec-
ion, wi h he dose ca e ully op imised, da a no shown) on
day 35. Blood samples we e collec ed on day 3 pos in ec ion
(p.i.; 1:1 a io wi h 12 mmol EDTA). NOD mice we e killed
on day 3 p.i. and panc eases sa ed o i us quan i ica ion and
his ological analysis. SOCS1- g mice we e moni o ed un il
diabe es de elopmen o day 21 p.i. and panc eases sa ed
o his ological analysis.
Moni o ing o blood glucose and diabe es de elopmen
Blood glucose was measu ed in blood ob ained om he
ail- ein wi h a Baye Con ou XT blood glucose me e
(Baye , Basel, Swi ze land) and a glucose concen a ion
>18 mmol/l, o wo consecu i e measu emen s be ween
13 and 18 mmol/l we e used o de ine diabe es and dia-
be ic mice we e killed.
Neu alising an ibody de ec ion Neu alising an ibody i es
we e de e mined by s anda d i us plaque educ ion assay in
g een monkey kidney (GMK) cells (Na ional Ins i u e o
Heal h and Wel a e, Finland; mycoplasma nega i e) [3].
Plaque numbe educ ion ≥80% compa ed wi h un ea ed
i us suspension was conside ed posi i e. The de ec ion limi
o he assay was a ou old dilu ion (1:4), and posi i i y cu -o
o se um samples was se o ≥1:16.
Vi us i a ion Panc eases we e homogenised and ly ic i us
quan i ied in ei he blood o panc eas by s anda d plaque as-
say in GMK cells. Vi al i es a e exp essed as PFU/g o issue
o ml o blood. See ESM Me hods: Vi us i a ion and issue
homogenisa ion o mo e de ails.
PCR analysis En e o i us speci ic eal- ime PCR was pe -
o med using RNA ex ac ed om blood samples; see ESM
Diabe ologia (2018) 61:476–481 477
Me hods: PCR analysis and Honkanen e al [8] o u he
de ails. P ime s a e shown in ESM Table 2.
His ological analysis and immunohis ochemis y
His ological analysis and immunohis ochemis y o panc eas
samples wi h i al capsid p o ein 1 (VP1), insulin and gluca-
gon we e ca ied ou as in Flods öm e al and La sson e al ([4,
6]; ESM Me hods: Immunohis ochemis y and an ibodies).
S a is ical analysis S a is ical analyses we e execu ed using
P ism 5 so wa e (G aphPad, La Jolla, CA, USA). PCR and
VP1 immunohis ochemis y da a we e analysed by χ
2
es s.
Plaque assay i us i a ions we e analysed by Mann–Whi ney
U es . Neu alising an ibody da a was analysed by one-way
ANOVA. Diabe es incidence was analysed by log ank
Man el–Cox es . Da a a e exp essed as he mean ± SD. A p
alue ≤0.05 was ega ded s a is ically signi ican .
Resul s
CVB1 accine is well ole a ed and is highly immunogenic
The newly p oduced CVB1 accine was well ole a ed by
NOD mice wi h no ad e se e ec s on weigh o blood glucose
(ESM Fig. 1a–c). Fu he mo e, he accine was highly immu-
nogenic and accina ed mice p oduced CVB1 neu alising
an ibodies a e he p ima y immunisa ion, which was aug-
men ed a e he second immunisa ion (ESM Fig. 1d).
Se um wi h a neu alising capaci y was no de ec ed in
bu e - ea ed mice (da a no shown).
CVB1 accine p o ec s agains CVB1 in ec ion in NOD
mice We nex examined whe he he accine p o ec s agains
i aemia caused by CVB1 in ec ion and p e en s i us epli-
ca ion in he panc eas on day 3 p.i. All accina ed mice (8/8)
we e p o ec ed om i aemia, as de e mined by RT-PCR and
Fig. 1 A CVB1 accine p o ec s NOD mice agains i aemia and sys-
emic i al sp ead ollowing in ec ion wi h CVB1. (a) Schema ic illus-
a ing he expe imen al imeline; NOD mice and SOCS1- g mice we e
accina ed o gi en bu e alone, ollowed by in ec ion wi h CVB1. Fo
NOD mice, he expe imen s we e e mina ed on day 3 p.i. Mock acci-
na ions and CVB1 accina ions a e ep esen ed by he emp y sy inge;
CVB1 challenge is shown wi h he black sy inge. (b)Pe cen ageo in-
ec ed accina ed (n= 8) o bu e - ea ed (n= 6) mice de e mined by he
p esence o CVB1 RNA in he blood o NOD mice on day 3 p.i. as
de ec ed by RT-PCR. ***p<0.001,χ
2
es . (c) Cy opa hic i us in he
blood and (d) in he panc eas on day 3 p.i. in NOD mice ea ed wi h
bu e (n=6)o CVB1 accine(n=8)asmeasu edbys anda dplaque
assay. Mean alues ± SD; **p< 0.01 and ***p<0.001,Mann–Whi ney
U es . (e) Rep esen a i e images o VP1 posi i i y (b own s aining) in
panc eas sec ions o bu e - ea ed and ( ) CVB1- accina ed NOD mice
on day 3 p.i. (×16 magni ica ion; scale ba , 50 μm) and (g) pe cen age o
mice wi h VP1 posi i i y in he panc eas. ***p<0.001,χ
2
es
478 Diabe ologia (2018) 61:476–481
plaque assay (Fig. 1b, c). Con e sely, all bu e - ea ed mice
we e iden i ied i aemic by plaque assay (Fig. 1c) and 5/6
we e posi i e o CVB1 RNA (Fig. 1b). Simila ly, eplica ing
i us in he panc eas was measu ed in bu e - ea ed mice bu
no in accina ed mice (Fig. 1d). Immunohis ochemical ana-
lysis using he VP1 an ibody u he con i med i al dissem-
ina ion o he panc eas in all bu e - ea ed mice (Fig. 1e, g)
bu no accina ed mice (Fig. 1 –g).
CVB1 accine p o ec s agains i us-induced diabe es
SOCS1- g mice, which a e suscep ible o i us-induced diabe-
es [4], showed no ad e se changes in weigh and blood glu-
cose a e CVB1 accina ion (da a no shown). Addi ionally,
accina ed SOCS1- g mice de eloped a obus an ibody e-
sponse (Fig. 2a) simila o ha obse ed in NOD mice
(ESM Fig. 1d). Bu e - ea ed mice emained consis en ly
nega i e o neu alising an ibodies (da a no shown).
Fig. 2 SOCS1- g mice a e p o ec ed om i us-induced diabe es by he
CVB1 accine. (a) Neu alising an ibody i es in he se um o accina ed
mice (n= 7) sampled p io o accina ion on days 0, 14 and 28 and be o e
in ec ion on day 35. The do ed line illus a es he neu alising capaci y
h eshold in he i us neu alisa ion assay. Each se um sample was
analysed in wo independen neu alisa ion assays and he mean
neu alising an ibody i e calcula ed. Mean alues a e indica ed by he
line ± SD; ***p< 0.001 compa ed wi h day 0 o indica ed ime poin as
de e mined by one-way ANOVA. (b,c) Weigh changes o indi idual
mice ea ed wi h accine bu e (n=6)(b), o CVB1 accine (n=7)
(c) a e in ec ion wi h 10
6
PFU CVB1. Each indi idual animal is ep e-
sen ed by a single line. Th ee o he bu e - ea ed animals de eloped
diabe es and we e emo ed p io o day 21. (d) Pe cen age o bu e -
ea ed (n= 6) o accina ed mice (n= 7) posi i e o CVB1 in he blood
on day 3 p.i. as de ec ed by RT-PCR. ***p<0.001,χ
2
es . (e)
Cy opa hic i us measu ed in he blood o bu e - ea ed (n=6)o ac-
cina ed (n= 7) mice on day 3 p.i. by s anda d plaque assay. Mean alues ±
SD; **p<0.01,Mann–Whi ney U es . ( ) Cumula i e diabe es incidence
in bu e - ea ed (black line) and accina ed (do ed line) SOCS1- g mice
a e in ec ion wi h CVB1, p< 0.05 compa ing he wo g oups as de e -
mined by log ank Man el–Cox es . Fo malin- ixed, pa a in embedded
SOCS1- g mice panc eas sec ions s ained wi h insulin o glucagon an i-
bodies by immunohis ochemis y. Shown a e ep esen a i e images om
(g) bu e - ea ed and (h) CVB1- accina ed mice. Images on he le o
each panel a e a ×16 magni ica ion and he whi e box indica es he a ea
o magni ica ion shown in he igh panels (a ×40 magni ica ion). Scale
ba s, 50 μm. (g) No e he loss o acina issue and immune cell in il a ion
in issue om bu e - ea ed animals
Diabe ologia (2018) 61:476–481 479
We nex moni o ed SOCS1- g mice a e CVB1 challenge.
No ob ious di e ences we e ound in he weigh o accina -
ed and bu e - ea ed mice (Fig. 2b, c). Fu he mo e, i aemia
measu emen s on day 3 p.i. e ealed no signs o in ec ion in
he accina ed animals (0/7; Fig. 2d, e). In con as , all (6/6)
bu e - ea ed mice we e in ec ed as indica ed by he de ec ion
o bo h i al RNA (Fig. 2d) and in ec i e i us by plaque
assay (Fig. 2e).
We also acked diabe es de elopmen in he in ec ed
SOCS1- g mice un il day 21 p.i. As expec ed, diabe es oc-
cu ed in he bu e - ea ed SOCS1- g mice wi h 50% (3/6)
de eloping hype glycaemia (p< 0.05; Fig. 2 ). Panc ea ic
exoc ine damage was no able in 4/6 mice (Fig. 2g), which
co esponded wi h diabe es de elopmen . Mo eo e , mice ha
de eloped hype glycaemia showed glucagon posi i i y bu a
loss o insulin posi i i y in a numbe o isle s, indica ing de-
s uc ion o he insulin-p oducing be a cells (Fig. 2g). In con-
as , all se en accina ed SOCS1- g mice we e p o ec ed om
diabe es (Fig. 2h) and showed no mal panc eas mo phology
on day 21 p.i. wi h heal hy exoc ine issue and in ense insulin
and glucagon s aining in he isle s o Lange hans (Fig. 2h).
Discussion
In he p esen s udy, we show ha a mono alen , o malin-
inac i a ed and non-adju an ed CVB1 accine p o ec s
agains bo h acu e CVB1 in ec ion and i us-induced diabe es
in a mouse model o i us-induced diabe es. The accine
p o ed o be highly immunogenic, wi h he an ibody i es
p oduced being g ea e han hose conside ed o be p o ec i e
in o he en e o i us accines [9] and was well ole a ed wi h
ega ds o weigh and blood glucose. Combined, hese esul s
highligh he po en ial o en e o i us accines in es ing he
hypo hesis ha p e en ing en e o i us in ec ions a enua es
he isk o ype 1 diabe es.
When conside ing en e o i us accine de elopmen o
clinical in e en ion ials, i is pe inen o iden i y en e o i-
uses wi h possible oles in ype 1 diabe es pa hogenesis.
La ge-scale p ospec i e s udies including he Type 1
Diabe es P edic ion And P e en ion P ojec (DIPP) and The
En i onmen al De e minan s o Diabe es in he Young
(TEDDY) S udy [2,10] a e he e o e highly impo an owing
o hei po en ial in he iden i ica ion o diabe ogenic i uses
om clinical samples collec ed. Mo eo e , i an en e o i us
accine we e success ully app o ed o clinical use, p ospec-
i e s udies like hese would p o ide excellen oppo uni ies o
es accine e icacy in he p e en ion o ype 1 diabe es.
Theo e ically, adi ional o malin-inac i a ed accines could
include se e al di e en en e o i us ypes. Fo example, polio
accine con ains polio i us 1-3 and ecen ly, a 50- alen hi-
no i us accine (bo h en e o i uses) had high immunogen-
ici y in nonhuman p ima es [11]. Howe e , he economic
easibili y o comme cial accine p oduc ion cu en ly limi s
he numbe o se o ypes ha can be included in a single ac-
cine. Exis ing in o ma ion sugges s he impo ance o CVB
en e o i uses as a ge s o accines o use in ype 1 diabe es
p e en ion ials and u u e s udies should aim o p oduce a
hexa alen accine including all six CVB se o ypes [2].
Mo eo e , his ype o accine would be aluable in he p e-
en ion o o he po en ially a al CVB associa ed diseases,
including myoca di is and asep ic meningi is.
In conclusion, his p oo o concep s udy demons a es ha
a o malin-inac i a ed CVB accine p o ec s agains i us-
induced diabe es. This p o ides a model o u u e de elopmen
o en e o i us accines, pa icula ly mul i alen accines co -
e ing a numbe o se o ypes o es ing in clinical ials o ex-
amine hei abili y o p e en human ype 1 diabe es.
Acknowledgemen s We would like o hank membe s o he he animal
acili y a Ka olinska Ins i u e (PKL, S ockholm, Sweden), U. Kiiskinen,
N. Kähkönen, M. Jokela, A. Ka jalainen, M. Kekäläinen, M. O askainen,
E. Jalonen, J. Mää ä, J. Saa ike u, N. Saa inen and L. Azizi (Facul y o
Medicine and Li e Sciences, Uni e si y o Tampe e, Finland) o hei
echnical suppo . We would also like o hank membe s o he Flods öm-
Tullbe g g oup (Depa men o Medicine, Ka olinska Ins i u e , Sweden)
o discussions and suppo . This wo k was p esen ed in a pos e o ma a
he JDRF nPOD 9 h Annual Scien i ic Mee ing in 2017.
Da a a ailabili y The da ase s gene a ed du ing and/o analysed du ing
he cu en s udy a e a ailable om he co esponding au ho on eason-
able eques .
Funding We would like o acknowledge TEKES (The Finnish Funding
Agency o Inno a ion: THERDIAB p ojec , dia y no. 1843/31/2014);
he Reino Lah ika i Founda ion, Finland; he Sig id Juselius Founda ion,
Finland; he Swedish Child Diabe es Founda ion; he Swedish Diabe es
Resea ch Associa ion and Ka olinska Ins i u e including he S a egic
Resea ch P og am in Diabe es, Sweden o inancial suppo . We also
hank he Ka olinska Uni e si y Hospi al and Biocen e Finland o in-
as uc u e suppo . Vac ech L d., A cDia L d., JILAB L d. and Finnmedi
Resea ch L d. a e acknowledged o hei con ibu ion wi hin
THERDIAB. The s udy sponso s we e no in ol ed in he design o he
s udy; he collec ion, analysis, and in e p e a ion o da a; w i ing he e-
po ; o he decision o submi he epo o publica ion.
Duali y o in e es HH is a mino (5%) sha eholde and membe o he
boa d o Vac ech L d., which de elops accines agains pico na i uses.
The o he au ho s decla e ha he e is no duali y o in e es associa ed
wi h hei con ibu ion o his manusc ip .
Con ibu ion s a emen MFT and VPH a e he gua an o s o his wo k
and, as such, had ull access o all o he da a in he s udy and ake
esponsibili y o he in eg i y o he da a and he accu acy o he da a
analysis. VMS planned expe imen s, gene a ed, analysed and in e p e ed
da a and w o e he manusc ip . MMH planned expe imen s, gene a ed,
analysed and in e p e ed da a and edi ed he manusc ip . ES and SO
gene a ed and analysed da a and edi ed he manusc ip . ASK con ibu ed
o discussions, analysed da a and edi ed he manusc ip . HH, OHL and
VPH con ibu ed o planning and discussions and edi ed he manusc ip .
MFT planned he s udy and expe imen s, analysed and in e p e ed da a
and edi ed he manusc ip . All au ho s app o ed he inal e sion o he
manusc ip .
480 Diabe ologia (2018) 61:476–481

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