Immune modulator adamantylamide dipeptide stimulates efficient major histocompatibility complex class I-restricted responses in mice.

CLINICAL AND VACCINE IMMUNOLOGY, May 2007, p. 538–543 Vol. 14, No. 5 1556-6811/07/$08.00⫹0 doi:10.1128/CVI.00316-06 Copy igh © 2007, Ame ican Socie y o Mic obiology. All Righ s Rese ed. Immune Modula o Adaman ylamide Dipep ide S imula es E icien Majo His ocompa ibili y Complex Class I-Res ic ed Responses in Mice 䌤 Pablo D. Becke , 1,2 Mi iam No¨ de , 1 Ca los A. Guzma´n, 1 * and Saul G ins ein 2 * Depa men o Vaccinology, Helmhol z Cen e o In ec ion Resea ch, Inho ens aße 7, D-38124 B aunschweig, Ge many, 1 and Labo a o io de Vi ologı´a, Hospi al de Nin˜os Rica do Gu ie´ ez, Gallo 1330, 1425 Buenos Ai es, A gen ina 2 Recei ed 23 Augus 2006/Re u ned o modi ica ion 16 Oc obe 2006/Accep ed 22 Feb ua y 2007 Adaman ylamide L-alanyl-D-isoglu amine (AdDP) is a syn he ic adju an which belongs o he amily o he desmu amyl pep ides. AdDP exe s i s adju an p ope ies when i is adminis e ed ei he by he pa en e al o by he mucosal ou e, leading o he elici a ion o s ong humo al esponses a bo h he sys emic and he mucosal le els. Howe e , e y li le is known abou he e ec o AdDP on cellula immuni y. He e we demon- s a e ha AdDP is able o s imula e cellula esponses, which a e cha ac e ized by he elease o gamma in e e on by CD8 ⴙ T cells when hey a e es imula ed wi h a majo his ocompa ibili y complex class I- e- s ic ed pep ide and s ong in i o lymphocy e-media ed cy o oxic ac i i y. The capaci y o AdDP o s imula e he elici a ion o bo h cellula and humo al adap i e esponses makes his adju an a p omising ool o he de elopmen o mucosal accine o mula ions. Mu amyl dipep ide (MDP; N-ace ylmu amyl-L-alanyl-D-iso- glu amine) is a syn he ic de i a i e o a componen p esen in cell wall pep idoglycans o many bac e ia. I is also he minimal bioac i e s uc u e equi ed o eplace he whole mycobac e ia p esen in he F eund’s comple e adju an . Expe imen al s ud- ies ha e demons a ed ha he immunomodula o y p ope ies o MDP and ce ain analogues, alone o in combina ion wi h o he agen s, can con e esis ance agains i uses (e.g., human immunode iciency i us, in luenza i us, he pes simplex i us, Sendai i us, Semliki Fo es i us, accinia i us, and mu ine hepa i is i us), bac e ia, and ungi (8, 12, 14, 19, 20). Howe e , he py ogenic and a h i ogenic e ec s o MDP p eclude i s use in humans (2). In o de o ake ad an age o i s immuno- modula o y p ope ies bu o minimize he isk o side e ec s, non oxic MDP de i a i es ha e been gene a ed, such as he adaman ylamide dipep ide (AdDP), MDP-Lys(L18), mu- abu ide (es e de i a e), and glucosaminylmu amyl dipep ide (1, 18). In he 1980s, a emp s o design new ools agains he in lu- enza i us led o he combina ion o aman adine, which has been ex ensi ely employed o he p ophylaxis and chemo he - apy o in luenza (4), wi h he dipep ide om MDP. The new compound no only e ains he an i i al cha ac e is ics o aman adine bu also possesses immunomodula o y p ope ies ha migh be desi able in a accine o mula ion. In ac , his no el syn he ic compound, called AdDP, was success ully es ed in p eclinical s udies alone o in combina ion wi h in- luenza i us an igens (21). Recen s udies ha e demons a ed ha AdDP is a powe ul adju an which is able o enhance humo al immune esponses agains coadminis e ed an igens a bo h he sys emic and he mucosal le els in di e en animal species (2). Addi ional wo k showed ha a accine o mula ion based on he ecombinan P6 p o ein and AdDP was able o con e p o ec ion agains pulmona y and middle ea in ec ions caused by non ypeable Haemophilus in luenzae (5). Howe e , he e ec s o AdDP and o he desmu amyl pep ides on cellula esponses a e poo ly cha ac e ized, i hey ha e any e ec s a all. Finally, he e is s ill agmen a y in o ma ion and a lack o consensus conce n- ing he gene al mechanisms o ac ion o his amily o adju- an s. Thus, he majo aim o his wo k was o p o ide insigh s on he in i o immunomodula o y p ope ies o AdDP, pa ic- ula ly a he le el o s imula ion o cellula immune esponses. The esul s ob ained demons a ed ha AdDP is able o s im- ula e he elease o gamma in e e on (IFN-␥)byCD8 ⫹ T cells and lymphocy e-media ed cy o oxic ac i i y in i o. MATERIALS AND METHODS An igens, pep ides and adju an . The model an igens ␤-galac osidase (␤-Gal) and o albumin (OVA) we e pu chased om Boeh inge (Mannheim, Ge many) and Sigma, espec i ely. The pep ides encompassing he majo his ocompa ibili y complex (MHC) class I immunodominan L d - es ic ed ␤-Gal pep ide (TPHPA RIGL) and k b - es ic ed OVA pep ide (SIINFEKL) (11, 15, 23) we e syn he- sized a he Helmhol z Cen e o In ec ion Resea ch (B aunschweig, Ge many). AdDP was syn hesized by Bachem, Swi ze land, unde good manu ac u ing p ac- ice guidelines by a p e iously desc ibed p ocedu e (10). Animals and cell cul u es. Female BALB/c (H-2 d ) and C57BL/6 mice (ages, 6 o 8 weeks) we e pu chased om Ha lam-Winkelmann GmbH (Bo chen, Ge - many) and we e ea ed in acco dance wi h local and Eu opean Communi y guidelines. Spleen cells we e g own in RPMI 1640 supplemen ed wi h 10% e al cal se um, 100 U/ml o penicillin, 50 ␮g/ml o s ep omycin, 5 ⫻10 ⫺5 M 2-me cap oe hanol, and 1 mM L-glu amine (GIBCO BRL, Ka ls uhe, Ge many) and we e main ained a 37°C in a humidi ied 5% CO 2 a mosphe e. Immuniza ion p o ocols. G oups o i e BALB/c mice each we e immunized by he in ape i oneal ou e h ee imes on days 1, 7, and 14 wi h ei he 50 ␮go * Co esponding au ho . Mailing add ess o Ca los A. Guzma´n: Depa men o Vaccinology, Helmhol z Cen e o In ec ion Re- sea ch, Inho ens a␤e 7, D-38124 B aunschweig, Ge many. Phone: 49 531 6181 4600. Fax: 49 531 6181 4699. E-mail: ca los.guzman @helmhol z-hzi.de. Mailing add ess o Saul G ins ein: Labo a o io de Vi ologı´a, Hospi al de Nin˜os Rica do Gu ie´ ez, Gallo 1330, 1425 Buenos Ai es, A gen ina. Phone: 54 11 4964 3118. Fax: 54 11 4962 6770. E-mail: [email p o ec ed]. 䌤 Published ahead o p in on 7 Ma ch 2007. 538 on Feb ua y 15, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://c i.asm.o g/Downloaded om ␤-Gal alone o ␤-Gal coadminis e ed wi h 200 ␮g o AdDP. Simila ly, C57BL/6 mice we e immunized by he in anasal ou e wi h 50 ␮g o OVA alone o OVA coadminis e ed wi h 100 ␮g o AdDP. Nega i e con ols ecei ed phospha e- bu e ed saline (PBS). Se um samples we e collec ed om blood o he ail ein 1 day be o e each immuniza ion and 1 week a e he las immuniza ion, when he mice we e killed by CO 2 inhala ion. Se a we e s o ed a ⫺20°C p io o de e mina ion o speci ic an ibodies. B onchoal eola la age luid samples we e ob ained by lushing he o gans wi h PBS supplemen ed wi h 10 mM phenyl- me hylsul onyl luo ide. The spleens we e emo ed asep ically and we e pooled o analysis o cellula immune esponses. Bone ma ow (BM) cells we e isola ed om he emu and ibia, as desc ibed elsewhe e (9). B ie ly, he legs we e sepa a ed, all muscle issues we e emo ed om he emu and ibia, and he epiphyses we e cu o . The bone ends we e punc u ed wi h a needle and he BM was lushed ou wi h medium. The concen a ion o he cells was adjus ed acco ding o he speci ic expe imen . De ec ion o speci ic an ibodies. ␤-Gal- and OVA-speci ic an ibodies we e de e mined by enzyme-linked immunoso ben assay (ELISA). B ie ly, 96-well Immuno MaxiSo p assay pla es (Nunc, Roskilde, Denma k) we e coa ed o e - nigh a 4°C wi h 100 ␮l o ei he ␤-Gal o OVA a 5 ␮g/ml in ca bona e bu e (pH 9.6). A e he wells we e blocked, he pla es we e washed and u he incuba ed wi h 100 ␮l o se ial wo old dilu ions o se a o 1ha 37°C. A e ou washes, de ec ion an ibody, ei he bio inyla ed ␥-o ␮-chain-speci ic goa an i- mouse an ibody (Sigma Chemie, Deisenho en, Ge many), was added. The pla es we e u he incuba ed o 1ha 37°C. A e he pla es we e washed, pe oxidase- conjuga ed s ep a idin (Pha Mingen) was added and he pla es we e incuba ed a oom empe a u e o 45 min. A e ano he ou washes, he eac ions we e de eloped by using 2,2⬘-azinobis(3-e hylbenz hiazoline-6-sul onic acid) in 0.1 M ci a e-phospha e bu e (pH 4.35) con aining 0.01% H 2 O 2 , and he abso bance was ead a a wa eleng h o 405 nm. The immunoglobulin G (IgG) iso ypes p esen in he se um samples we e de e mined by an ELISA, as desc ibed p e iously (2), by using as seconda y an ibodies bio in-conjuga ed a an i-mouse IgG1, IgG2a, IgG2b, o IgG3 (Sou he n Bio echnology Associa es, Bi mingham, AL). The abso bance alues we e plo ed agains he dilu ions, and he endpoin i e s we e de e mined as he highes sample dilu ion ha ga e an A 405 o ⱖ0.2 abo e he backg ound alues. The esul s we e exp essed as mean ⫾s anda d e o s o he means (SEMs) o each g oup. The p esence o OVA-speci ic sec e o y IgA in lung la age luid specimens was de e mined as desc ibed p e- iously (3). De ec ion o Ig-p oducing cells by ELISPOT assay. The numbe s o o al and an igen-speci ic IgG sec e ing cells we e de e mined by enzyme-linked immuno- spo (ELISPOT) assay. B ie ly, poly inylidene di luo ide pla es (Millipo e, Bed- o d, MA) we e coa ed wi h ei he 100 ␮l/well o iso ype-speci ic cap u e an i- bodies (Sigma, Ge many) a a concen a ion o 5 ␮g/ml o an igen (␤-Gal o OVA a 5 ␮g/ml in ca bona e bu e , pH 9.6) o de e mine o al and an igen- speci ic an ibodies, espec i ely. Di e en concen a ions o BM cells we e in- cuba ed in quad uplica e o 6 h. Then, he pla es we e washed, 100 ␮lo he co esponding bio inyla ed de ec ion an ibody (Sigma) was added, and he pla es we e u he incuba ed o e nigh a 4°C. A e se e al washes, he pla es we e incuba ed o 1 h wi h 100 ␮l/well o pe oxidase-conjuga ed s ep a idin (BD- Pha mingen, Ge many). The spo s we e de eloped by using 3-amino-9-e hylca - bazole (Sigma) in 0.1 M ace a e bu e (pH 5.0) and 0.05% H 2 O 2 (30%). The eac ion was s opped by insing he pla es wi h ap wa e , and he pla es we e ai d ied. The spo s we e scanned wi h an ImmunoSpo se ies 3A analyze and we e coun ed by using ImmunoSpo image analyze so wa e ( e sion 3.2; Cellula Technology, L d.). De ec ion o IFN-␥- and IL-4-p oducing cells by ELISPOT assay. The numbe o IFN-␥and in e leukin-4 (IL-4)-sec e ing cells was de e mined by ELISPOT assay, acco ding o he manu ac u e ’s ins uc ions (Bec on Dickinson). In b ie , spleen cells we e added a inal concen a ions o 1 ⫻10 6 and 5 ⫻10 5 cells/well and we e incuba ed in quad uplica e in he absence o p esence o di e en concen a ions o he MHC class I- es ic ed pep ide (IFN-␥) o OVA (IL-4). A e 16 h o cul u e, he cells we e emo ed and locally p oduced single-cell- de i ed IFN-␥o IL-4 was de ec ed by using IFN-␥o IL-4 bio inyla ed an i- bodies, which we e de eloped by addi ion o pe oxidase-conjuga ed s ep a idin and subs a es, as desc ibed abo e. Measu emen o cellula p oli e a ion. P oli e a ion assays we e pe o med in iplica e, as desc ibed p e iously (6). B ie ly, spleen cells (5 ⫻10 5 cells/well) we e incuba ed o 4 days in he p esence o ␤-Gal o OVA. Eigh een hou s be o e ha es , 1 ␮Ci o [ 3 H] hymidine (Ame sham In e na ional, F eibu g, Ge many) was added o each well. The cells we e ha es ed on il e pape (Fil e ma A; Wallac, F eibu g, Ge many) by using a cell ha es e (Ino ech, Wohlen, Swi ze land), and he amoun o [ 3 H] hymidine inco po a ed in o he DNA o p oli e a ing cells was de e mined wi h a ␥-scin illa ion coun e (Wallac 1450; Mic o-T ilux). De e mina ion o lymphocy e-media ed cy o oxi y in i o. Suspensions o splenocy es om naı¨ e C57BL/6 mice we e deple ed o ed cells and spli in o wo equal po ions. One po ion was labeled wi h a high concen a ion (1 ␮M) o ca boxy luo escein-succinyl-es e (CFSE hi ; Molecula P obes) and pulsed o 1 h a 37°C wi h he dominan OVA pep ide (amino acids 257 o 264) a a concen a ion o 15 ␮g/ml. The o he po ion was labeled wi h a low concen a- ion (0.1 ␮M) o CFSE (CFSE lo ) and was u he incuba ed o 1ha 37°C wi hou pep ide. Equal numbe s o each cell popula ion we e mixed. A o al amoun o 2 ⫻10 7 cells was adop i ely ans e ed by in a enous injec ion in o he immunized mice. Cells om he spleen we e analyzed by low cy ome y a e 16 h wi h a FACScalibu ins umen and BD Cell Ques P o so wa e. The pe cen OVA-speci ic lysis was de e mined by he loss o he pep ide-pulsed CFSE hi popula ion and compa ed o he con ol CFSE lo popula ion (13). The ollowing o mula was used o calcula e he pe cen age o speci ic lysis: 100 ⫺ {[(pe cen CFSE hi in immunized mice/pe cen CFSE lo in immunized mice)/ (pe cen CFSE hi in con ol mice/pe cen CFSE lo in con ol mice)] ⫻100}. S a is ical analysis. The s a is ical signi icance o he di e ences be ween wo g oups was de e mined om he means and he SEMs by he S uden ’s wo- ailed es , and he s a is ical signi icance o he di e ences be ween h ee o mo e g oups was de e mined by one-way analysis o a iance. Di e ences we e conside ed signi ican a a P alue o ⬍0.05. RESULTS Immuniza ion wi h AdDP as he adju an by ei he pa en- e al o mucosal ou e esul s in elici a ion o s ong an igen- speci ic an ibody esponses. Fi s , we e alua ed i he use o AdDP esul s in he s imula ion o s ong an ibody esponses in BALB/c mice ecei ing he model an igen ␤-Gal, as was p e iously demons a ed wi h o he immunogens, such as OVA. To his end, we measu ed he ␤-Gal-speci ic an ibody esponses be o e each immuniza ion and 1 week a e he las boos . In se um samples collec ed a day 6, 1 week a e he i s immuniza ion, a weak esponse o ␤-Gal-speci ic IgM an- ibodies was obse ed in he g oups ecei ing ei he ␤-Gal alone o ␤-Gal coadminis e ed wi h AdDP (Fig. 1). Howe e , a day 13, 1 week a e he second immuniza ion, he g oup ha ecei ed ␤-Gal oge he wi h AdDP showed six old highe i e s han he g oup immunized wi h ␤-Gal alone (Fig. 1). A he same ime poin , mice ecei ing AdDP as he adju an had ␤-Gal-speci ic IgG i e s ha we e also sligh ly di e en om he i e s in hose ecei ing ␤-Gal alone (Fig. 1). A day 21, animals ecei ing ␤-Gal coadminis e ed wi h AdDP showed 22- old highe an igen-speci ic IgM i e s han con ol animals immunized wi h ␤-Gal alone (P⬍0.01). In addi ion, he ␤-Gal-speci ic IgG an ibody i e s we e 16- old highe in mice accina ed wi h AdDP han in con ol mice ecei ing ␤-Gal (P⬍0.05) (Fig. 1). The subclass p o ile o he ␤-Gal- speci ic se um Igs was measu ed a he las ime poin . BALB/c mice immunized wi h ␤-Gal coadminis e ed wi h AdDP showed dominan speci ic IgG1 subclass an ibodies (da a no shown), which a e gene ally associa ed wi h a Th2- ype esponse (7, 24). In all p e ious wo k, BALB/c mice we e used o demon- s a e he po ency o AdDP as he adju an . Thus, we also used C57BL/6 mice in he p esen s udy o ensu e ha he immune esponses obse ed a e no es ic ed o he BALB/c s ain. The e o e, he model an igen, OVA, was coadminis e ed wi h AdDP o C57BL/6 mice by he in anasal ou e and he an i- body esponses we e e alua ed. C57BL/6 mice immunized wi h OVA plus AdDP showed s ong IgG p oduc ion in com- pa ison wi h ha o he animals ecei ing OVA alone (P⬍ VOL. 14, 2007 IN VIVO EFFECT OF AdDP 539 on Feb ua y 15, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://c i.asm.o g/Downloaded om 0.05) (Fig. 2). As expec ed, AdDP was also able o p omo e he elici a ion o a mucosal immune esponse in mice accina ed by he in anasal ou e. A 10- old inc emen in he le els o OVA-speci ic sec e o y IgA was obse ed in lung la age luid samples om animals o which OVA was coadminis e ed wi h AdDP compa ed o ha in mice ecei ing OVA alone. These esul s we e in ag eemen wi h he obse ed inc emen in he numbe o IL-4-p oducing cells (98 e sus 0 pe 10 6 spleno- cy es in he g oup ea ed wi h OVA plus AdDP and he con ol g oup, espec i ely) and he p esence o a dominan an i-OVA IgG1 esponse in se a. Immuniza ion wi h AdDP as adju an esul s in he accu- mula ion o an igen-speci ic Ig-sec e ing cells in BM. I is known ha a e p ima y con ac wi h an an igen, a sho an ibody esponse cha ac e ized by he expansion o an ibody- sec e ing plasmablas s is gene a ed. Howe e , mos an ibody- sec e ing cells (ASCs) p oduced du ing seconda y immune esponses lea e he seconda y lymphoid o gans, wi h he main inal des ina ions being BM, mucosa-associa ed issues, and ch onically in lamed issues (16). These ASCs wi h he pheno- ype o ma u e plasma cells ha e a po en ial li e span o mo e han 1 yea . Because he IgG subclass has a hal -li e o 3 weeks, he main enance o an ibody i e s o e a long pe iod is he esponsibili y o BM ASCs (22). The e o e, we examined he abili y o BM cells o sec e e o al and ␤-Gal-speci ic IgG a e 6 h o es imula ion by he ELISPOT assay. This sho incu- ba ion pe iod is no enough o he di e en ia ion o memo y B cells in o ASCs; hus, only ac i ely sec e ing plasma cells a e de e mined (17). We did no ind any s a is ically signi ican di e ence in he numbe o o al IgG-sec e ing cells be ween BALB/c mice ecei ing ␤-Gal alone o ␤-Gal in combina ion wi h AdDP (Fig. 3, le panel). In con as , we obse ed a signi ican (P⬍0.001) inc ease in he numbe o ␤-Gal-speci ic IgG-sec e ing cells in mice immunized wi h ␤-Gal oge he wi h AdDP in compa ison wi h he numbe in hose immu- nized wi h ␤-Gal alone (Fig. 3, igh panel). Six y days a e he las immuniza ion, C57BL/6 mice ha we e immunized wi h OVA plus AdDP also exhibi ed high i e s o an ibodies ha co ela ed wi h he numbe o o al OVA-speci ic IgG ASCs, wi h IgG1 being he majo IgG subclass p oduced, as de e - mined by he ELISPOT assay (da a no shown). These indings ag ee wi h ou p e ious esul s, whe ein we showed he main- enance o high speci ic IgG i e s o a pe iod o 1 yea (2). Immuniza ion wi h AdDP as adju an s imula es s ong cellula immune esponses. The cellula immune esponses induced in BALB/c mice we e e alua ed by assessing he p o- li e a i e capaci y o spleen cells a e in i o s imula ion wi h FIG. 1. Kine ic analysis o ␤-Gal-speci ic se um an ibodies in accina ed animals. BALB/c mice we e immunized wi h PBS, ␤-Gal (50 ␮g) alone, o ␤-Gal coadminis e ed wi h AdDP (200 ␮g) as he adju an . Immuniza ions a e indica ed by a ows (days 1, 8, and 15). An ibody i e s we e de e mined by endpoin dilu ion ELISA. Each poin ep esen s he g oup mean i e o o al an i-␤-Gal IgM (le panel) and IgG ( igh panel). The SEMs a e indica ed by e ical lines. Di e ences we e s a is ically signi ican a P⬍0.05 (*) and P⬍0.001 (**). FIG. 2. Analysis o OVA-speci ic se um an ibodies in C57BL/6 mice immunized by he in anasal (i.n.) ou e wi h AdDP as he adju- an . OVA-speci ic IgG i e s om se um samples we e de e mined by endpoin dilu ion ELISA. Each ba ep esen s he g oup mean end- poin i e . The SEMs a e indica ed by e ical lines. Di e ences we e s a is ically signi ican a P⬍0.05 (*). FIG. 3. De e mina ion o he numbe o an ibody-sec e ing cells p esen in immunized animals. Spleen cells om BALB/c mice acci- na ed wi h ␤-Gal we e cul u ed o 6 h, and he numbe o IgG- sec e ing cells was e alua ed by he ELISPOT assay. The esul s a e p esen ed as he o al numbe o IgG spo - o ming uni s (SFU)/10 6 cells (le panel) and ␤-Gal-speci ic IgG spo - o ming uni s/10 6 cells ( igh panel). The SEMs o quad uplica e alues a e indica ed by e ical lines. Di e ences we e s a is ically signi ican a P⬍0.0001 (***). 540 BECKER ET AL. CLIN.VACCINE IMMUNOL. on Feb ua y 15, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://c i.asm.o g/Downloaded om ␤-Gal. A dose-dependen p oli e a i e esponse was obse ed in mice immunized wi h ei he ␤-Gal alone o ␤-Gal coadmin- is e ed wi h AdDP bu no in con ol mice (Fig. 4). A sligh ly s onge esponse was obse ed in mice ecei ing ␤-Gal and AdDP compa ed o ha obse ed in hose ecei ing ␤-Gal alone, bu only a he highes concen a ion o ␤-Gal es ed (40 ␮l/ml). Howe e , he numbe o CD8 ⫹ IFN-␥-p oducing cells consis en ly inc eased in mice accina ed wi h AdDP as he adju an a e es imula ion wi h he pep ide encompassing he immunodominan MHC class I- es ic ed epi ope compa ed o he numbe o CD8 ⫹ IFN-␥-p oducing cells in hose ecei ing ␤-Gal alone (Fig. 5). The di e ence in he numbe o IFN-␥- sec e ing cells de ec ed a e in i o es imula ion in animals ecei ing ␤-Gal alone and ␤-Gal coadminis e ed wi h AdDP was s a is ically signi ican o e he ange o pep ide concen- a ions es ed ( o 2.5, 5, and 10 ␮g/ml, P⬍0.02, P⬍0.05, and P⬍0.005, espec i ely), wi h a maximum a 5 ␮g/ml (Fig. 5). Simila esul s we e ob ained when a simila analysis was pe - o med wi h BM cells and he pep ide a a concen a ion o 5 ␮g/ml (P⬍0.01) (Fig. 5). On he o he hand, C57BL/6 mice immunized wi h OVA plus AdDP by he in anasal ou e showed s onge in i o cy o oxic T-lymphocy e ac i i y han mice immunized wi h OVA alone (Fig. 6), sugges ing ha despi e he obse ed Th2-biased esponse pa e n, immuniza- ion wi h AdDP also induces a s ong Th1 componen . DISCUSSION Du ing ecen decades, he inc easing in e es in he de el- opmen o mucosal accines has led o he ac i e sea ch o new e ec i e adju an s. Se e al molecules om di e en mi- c oo ganisms ha e been p oposed o his pu pose. MDPs, bac e ial oxins (e.g., chole a oxin and Esche ichia coli hea - labile oxin), and Toll-like ecep o agonis s (e.g., CpG mo i s and MALP-2) ha e been among he mos e ec i e adju an s desc ibed up o now. Howe e , some o hem ha e shown side e ec s ha p eclude hei use in humans. In addi ion, o achie e op imal esul s ollowing accina ion, i would be nec- FIG. 4. P oli e a i e esponses s imula ed in immunized animals. The spleen cells o accina ed mice we e es imula ed wi h di e en concen a ions o ␤-Gal o 4 days. P oli e a ion was assessed by mea- su emen o [ 3 H] hymidine inco po a ion. The esul s a e exp essed as he a io be ween alues (a e age o iplica es) om s imula ed o nons imula ed samples (s imula ion index). SEMs a e indica ed by e ical lines. FIG. 5. S imula ion o IFN-␥-sec e ing cells in immunized animals. The numbe o IFN-␥-sec e ing cells was de e mined by he ELISPOT assay. Spleen cells we e incuba ed o 16 h in he p esence o he absence o a pep ide co esponding o he immunodominan L d - es ic ed ␤-Gal epi ope (TPHPARIGL), which is speci ic o MHC class I p esen a ion. The esul s a e p esen ed as he numbe s o IFN-␥spo - o ming uni s (SFU)/10 5 cells. The SEMs o quad uplica e alues a e indica ed by e ical lines. Di e ences we e s a is ically signi ican a P⬍0.05 (*), P⬍0.02 (**), P⬍0.01 (***), P⬍0.005 (§), and P⬍0.001 (§§). FIG. 6. Analysis o he lymphocy e-media ed cy o oxic ac i i y s imula ed in i o in C57BL/6 mice immunized wi h AdDP as he adju an . Spleen cells om naı¨ e C57BL/6 mice labeled wi h 0.1 ␮M and 1 ␮M CFSE we e incuba ed alone o wi h a pep ide encompassing he dominan MHC class I- es ic ed epi ope om OVA (SIINFEKL). A e he cells we e washed, equal numbe s o cells o each ype we e mixed. A o al o 2 ⫻10 7 cells we e adop i ely ans e ed by in a- enous injec ion in o immunized mice. A e 16 h, he spleens we e ha es ed and CFSE hi and CFSE lo cells we e de ec ed by low cy om- e y. Resul s a e exp essed as he pe cen age o speci ic lysis o pep- ide-pulsed cells. i.n., in anasal. VOL. 14, 2007 IN VIVO EFFECT OF AdDP 541 on Feb ua y 15, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://c i.asm.o g/Downloaded om essa y o s imula e di e en ypes o immune esponses, ac- co ding o he speci ic needs. I is ex emely unlikely ha his will be achie ed by he use o a single adju an molecule. Thus, he e is an u gen need o new adju an compounds. P e ious s udies sugges ed ha mu amyl pep ides and hei de i a i es a e p omising adju an s (18). Howe e , only a ew mu amyl pep ides, pa icula ly desmu amyl pep ides, ha e been cha ac e ized. We ha e p e iously shown ha an igen coadminis a ion wi h AdDP esul s in he s imula ion o s ong humo al esponses bo h a he sys emic le el and a he mucosal le el (2, 5). P eclinical s udies also demons a ed ha inco po a ion o AdDP in o a P6-based accine o mula ion esul ed in he s imula ion o a p o ec i e esponse agains non ypeable H. in luenzae (5). Howe e , up o now he e has been almos no knowledge abou he cellula immune e- sponses e oked by AdDP. Hence, we pe o med a ho ough cha ac e iza ion o he cellula immune esponses s imula ed by AdDP a e immuniza ion wi h he well-cha ac e ized model an igens ␤-Gal and OVA. In ag eemen wi h wha was p e iously epo ed, mice im- munized wi h AdDP as he adju an showed s ong humo al esponses. AdDP was able o enhance IgM p oduc ion and o induce he swi ch o IgG (3, 5). A signi ican inc emen in he numbe o an igen-speci ic ASCs was obse ed when spleno- cy es and BM cells we e es ed. Six y days a e immuniza ion wi h AdDP as he adju an , an igen-speci ic ASCs we e ound in BM. Because an ibody i e s a e main ained o long pe iods a e immuniza ion wi h AdDP and he IgG subclass has a hal -li e o 3 weeks, hese esul s p o ide an explana ion o ou p e ious indings. Thus, immuniza ion wi h AdDP as he adju an imp o es he mig a ion o plasmablas s om he ol- licles o seconda y lymph nodes o he BM and hei di e en- ia ion in plasma cells. Ano he impo an cha ac e is ic o an adju an is he abili y o induce a cellula esponse. In he case o mu amyl pep ides and de i a i es, mos o he s udies showed ha hey a e able o s imula e humo al immune esponses. Howe e , he esul s ob ained conce ning he de elopmen o cellula immune e- sponses a e ei he unclea o con adic o y (25). The e o e, a de ailed analysis o he cellula immune esponses s imula ed in he p esence o AdDP was pe o med. In acco dance wi h he obse ed IgG iso ype pa e n (i.e., dominan IgG1 e- sponse) and he p esence o IL-4-sec e ing cells, mice immu- nized wi h AdDP exhibi ed a majo Th2 esponse pa e n. Despi e he modes cellula p oli e a ion obse ed a e e- s imula ion in i o o cells om AdDP-immunized mice, s ong MHC class I- es ic ed esponses we e also p esen in hese animals. In ac , a signi ican inc emen in he numbe o IFN-␥-sec e ing cells, as well as a s ong in i o cy o oxic T- lymphocy e esponse, was obse ed in mice immunized by he in anasal ou e. Conside ing ou p e ious esul s and he new da a ha ha e eme ged om he p esen s udy, we conclude ha AdDP i is a p omising adju an able o s imula e no only he humo al immune esponse bu also s ong cellula immune esponses, which can be exploi ed o he de elopmen o new and mo e e icien accine o mula ions o p e en o ea human o e e ina y diseases. 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