A Coxsackievirus B Vaccine Protects Against Virus-Induced Diabetes in an Experimental Mouse Model for Type 1 Diabetes

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 . 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