Immunization of pigs to prevent disease in humans: construction and protective efficacy of a Salmonella enterica serovar Typhimurium live negative-marker vaccine.

INFECTION AND IMMUNITY, May 2007, p. 2476–2483 Vol. 75, No. 5 0019-9567/07/$08.00⫹0 doi:10.1128/IAI.01908-06 Copy igh © 2007, Ame ican Socie y o Mic obiology. All Righ s Rese ed. Immuniza ion o Pigs To P e en Disease in Humans: Cons uc ion and P o ec i e E icacy o a Salmonella en e ica Se o a Typhimu ium Li e Nega i e-Ma ke Vaccine 䌤 † Ma in Selke, 1 Jochen Meens, 1 S en Sp inge , 2 Ronald F ank, 3 and Ge ald-F. Ge lach 1 * Ins i u e o Mic obiology, Depa men o In ec ious Diseases, Uni e si y o Ve e ina y Medicine Hanno e , Hanno e , Ge many 1 ; Imp s o we k Dessau-To nau GmbH, PSF 214, Rodleben OT To nau, Ge many 2 ; and Depa men o Chemical Biology, Helmhol z Cen e o In ec ion Resea ch (HZI), Masche ode Weg 1, D-38124 B aunschweig, Ge many 3 Recei ed 4 Decembe 2006/Re u ned o modi ica ion 21 Janua y 2007/Accep ed 4 Feb ua y 2007 Zoono ic in ec ions caused by Salmonella en e ica se o a Typhimu ium pose a cons an h ea o consume heal h, wi h he pig being a pa icula ly majo sou ce o mul id ug- esis an isola es. Vacci- na ion, as a p omising app oach o educe coloniza ion and shedding, has been sca cely used, as i in e e es wi h cu en con ol p og ams elying on se ology as a means o he d classi ica ion. In o de o o e come his p oblem, we se ou o de elop a nega i e-ma ke accine allowing he di e en ia ion o in ec ed om accina ed animals (DIVA). Applying an immunop o eomic app oach wi h wo-dimensional gel elec opho esis, Wes e n blo , and quad upole ime-o - ligh andem mass spec ome y, we iden i ied he OmpD p o ein as a sui able nega i e ma ke . Using allelic exchange, we gene a ed an isogenic mu an o he licensed li e accine s ain Salmopo c and showed ha i ulence o Salmopo c and ha o he mu an s ain, Salmopo c⌬ompD, we e indis inguishable in BALB/c mice. In a pig in ec ion expe imen including wo o al immuniza ions wi h Salmopo c⌬ompD and challenge wi h a mul i esis an S.en e ica se o a Typhimu ium DT104 clinical isola e, we con i med he p o ec i e e icacy o Salmopo c⌬ompD in pigs, showing a signi ican educ ion o bo h clinical symp oms and coloniza ion o lymph nodes and in es inal ac . OmpD immunogenic epi opes we e de e mined by pep ide spo a ay analyses. Upon es ing o se e al 9-me pep ides, each including an immunogenic epi ope, one pep ide (posi ions F 100 o Y 108 ) ha acili a ed he de ec ion o in ec ed animals independen o hei accina ion s a us (DIVA unc ion) was iden i ied. The app oach desc ibed o e comes he p oblems cu en ly limi ing he use o bac e ial li e accines and holds conside able po en ial o u u e de elopmen s in he ield. Bac e ial ood-bo ne pa hogens a e o inc easing conce n o consume s and policy make s wo ldwide, as hey e- quen ly cause epidemic in es inal disease ou b eaks and he eby a e esponsible o high economic losses. One o he mos impo an ood-bo ne pa hogens is Salmonella en e ica se o a Typhimu ium, which is esponsible o 1.9% (Czech Republic) o 64.7% (New Zealand) o he non yphoid Sal- monella in ec ions epo ed wo ldwide o he yea s 2000 o 2001 (16). In he Uni ed S a es, non yphoidal Salmonella s ains a e esponsible o an es ima ed 1.41 million cases o human in ec ions and mo e han 500 human dea hs annually (24). In Eu ope, he numbe o o icially epo ed cases amoun s o mo e han 175,000 acco ding o he epo o he Eu opean Food Sa e y Au ho i y (EFSA) o he yea 2005 (h p://www.e sa.eu opa.eu/e c/medialib/e sa/science/moni o ing _zoonoses/ epo s/zoonoses_ epo _2005.Pa .0001.File.da /Zoonoses_ epo _2005.pd ), wi h he es ima ed numbe o un epo ed cases being se e al imes highe . The eme gence and apid sp ead o mul id ug esis an Sal- monella spp. (especially phage ype DT104) (1, 3, 30, 34) limi he he apeu ic al e na i es in cases o in asi e in ec ions and ha e been shown o be associa ed wi h an inc eased bu den o illness (16). Thus, in Eu ope, 9.1% o human Salmonella in ec- ions a e caused by S.en e ica se o a Typhimu ium, wi h 21.4% o hese in ec ions being due o phage ype DT104 (EFSA epo o 2005 [see abo e]). Mul id ug- esis an S.en e ica se o a Typhimu ium s ains a e ound in pigs wi h pa icula ly high equency (15) and can be isola ed om po k and po k p od- uc s (7, 21) (EFSA epo o 2005 [see abo e]). In Ge many, o example, 3.2% o samples aken om minced po k mea we e Salmonella posi i e: 67% o hese isola es we e S.en e ica se o a Typhimu ium, and 69% o hese we e esis an o mo e han ou an ibio ics (EFSA epo o 2005 [see abo e]). In addi ion, non- ood-bo ne animal- o-human (17, 51) and hu- man- o-human (33) ansmissions o sys emically sp eading mul id ug- esis an S.en e ica se o a Typhimu ium DT104 s ains ha e been epo ed, emphasizing he po en ial isk o his pa hogen o human heal h. In o de o educe he po en ial isk o consume s, i is c ucial o minimize pa hogen in ake in o he ood chain. In he Eu opean Union, su eillance sys ems a e equi ed ( egula ion [EC] 2160/2003; h p://eu opa.eu.in /eu -lex/p i/en/oj/da /2003 /l_325/l_32520031212en00010015.pd ), and a numbe o coun- ies a e speci ically moni o ing he occu ence o Salmonella in he po k p oduc ion chain (2, 11, 19, 45, 48). In pigs, Sal- * Co esponding au ho . Mailing add ess: Ins i u u¨ Mik obiologie, Zen um u¨ In ek ionsmedizin, S i ung Tie a¨ z liche Hochschule Han- no e , Bischo shole Damm 15, 30173 Hanno e , Ge many. Phone: 49 511 856 7598. Fax: 49 511 856 7697. E-mail: [email p o ec ed]. † Supplemen al ma e ial o his a icle may be ound a h p://iai .asm.o g/. 䌤 Published ahead o p in on 12 Feb ua y 2007. 2476 on Janua y 20, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://iai.asm.o g/Downloaded om monella moni o ing is based on mea juice se ology (26). The e o e, immuniza ion wi h con en ional accines canno be used as a means o educe in ec ion and shedding o he pa ho- gen, bu o mula ions o accines acili a ing he di e en ia ion o in ec ed and accina ed animals (DIVA) (4, 9, 46) would be equi ed. DIVA li e accines ha e been used wi h good success o he elimina ion o i al in ec ions such as Aujeszky’s disease in pigs (42, 43) o bo ine he pes i us in ec ions (46). Fo bac e ial diseases, only expe imen al DIVA li e accines ha e been de- sc ibed (22), and no DIVA accines agains ood-bo ne pa ho- gens ha e been cons uc ed o da e. This likely is due o he easy use o an ibio ics in li es ock o he apy and as eed addi i es un il he ecen pas (5, 47), he cos s and expe imen- al di icul ies in ol ed in bac e ial DIVA li e accine cons uc- ion, and, as gene ically modi ied o ganisms a e in ol ed, he necessi y o license hese accines a he Eu opean Agency o he E alua ion o Medicinal P oduc s. This licensing p oce- du e equi es ex ensi e expe imen al es ing and he e o e is p o i able only i he ma ke demand is su icien . He e, we p esen he de elopmen o an S.en e ica se o a Typhimu ium DIVA li e accine o pigs. We desc ibe a gen- e al app oach o cons uc and es a bac e ial DIVA li e accine in ol ing (i) he iden i ica ion a o nonessen ial immu- nogenic an igen by wo-dimensional polyac ylamide gel elec- opho esis (2D-PAGE), Wes e n blo ing, and quad upole ime-o - ligh (Q-TOF) andem mass spec ome y (MS/MS), (ii) he cons uc ion o an isogenic in- ame mu an lacking o eign DNA by allelic exchange, (iii) he de elopmen o a disc imina o y enzyme-linked immunoso ben assay (ELISA) upon epi ope mapping by pep ide spo a ay analyses, and (i ) an exempla y immuniza ion and challenge expe imen . MATERIALS AND METHODS Bac e ial s ains, plasmids, and p ime s. The bac e ial s ains, plasmids, and p ime s used in his wo k a e lis ed in Table 1. P epa a ion o ou e memb ane-associa ed p o eins. Memb ane-associa ed p o eins we e p epa ed as desc ibed p e iously (14). B ie ly, S.en e ica se o a Typhimu ium cells we e g own wi h shaking in 150 ml o Lu ia-Be ani (LB) b o h a 37°C o an op ical densi y a 600 nm (OD 600 ) o 0.7. Cells we e ha es ed by cen i uga ion a 8,000 ⫻g o 10 min a 4°C, esuspended in 30 ml o de e gen wash bu e (NaCl [150 mM], T is-HCl [10 mM, pH 8.0], sodium deoxychola e [0.05%], and sodium azide [0.04%]), incuba ed o 30 min in a shaking incuba o a 37°C, and cen i uged o 10 min a 7,000 ⫻g. This ea - men e icien ly solubilizes memb ane-associa ed lipop o eins while lea ing he cells mos ly in ac . The cell- ee supe na an con aining ou e memb ane-asso- cia ed p o eins as well as con amina ing in eg al ou e memb ane p o eins was il e ed (0.22-␮m po e size) and s o ed a ⫺20°C un il u he use. P o ein concen a ions we e de e mined by Mic o-BCA (Pie ce Bio echnology, Rock- o d, ILL). Fo 2D-PAGE, he cell- ee supe na an was p ecipi a ed o e nigh wi h ichlo oace ic acid (10% inal concen a ion). A e cen i uga ion a 15,000 ⫻g o 15 min a 4°C, pelle s we e washed wice wi h pu e ace one, esuspended in 500 o 1,000 ␮l bidis illed wa e , and s o ed a ⫺20°C. P epa a ion o ou e memb ane p o eins. Bac e ia g own and ha es ed as desc ibed abo e we e esuspended in 2 ml T is-HCl (50 mM, pH 8.0) wi h suc ose (25%, w / ol) and ozen a ⫺70°C. A e hawing, bac e ia we e incu- ba ed wi h lysozyme (2 mg/ml) and sa cosyl (2%, w / ol) o 1 h, ollowed by sonica ion (B anson-Soni ie B-30; Heinemann, Schwa¨bisch Gmu¨nd, Ge many) using a mic o ip a he maximum-s eng h se ing o h ee cycles o 30 s and a du y cycle o 60%. The lysa e was cen i uged (15,000 ⫻g o 30 min), ollowed by ul acen i uga ion o he supe na an (100,000 ⫻g o 90 min). The sonica- ion in combina ion wi h sa cosyl disin eg a es he cy oplasmic memb ane and emo es ou e memb ane-associa ed p o eins. The esul ing pelle con aining he ou e memb ane ac ion wi h in eg al memb ane p o eins was esuspended in 100 ␮lH 2 O and s o ed a ⫺20°C. De ec ion o immunogenic ou e memb ane-associa ed p o eins. Aliquo s o 500 ␮g o su ace-associa ed p o eins we e sepa a ed using 2D-PAGE wi h ImmobilineD yS ips (24 cm, pH 4 o 7). Sepa a ed p o eins we e ans e ed on o a ni ocellulose memb ane using a semid y-p o ein ans e sys em (No aBlo ; Ame sham Pha macia Bio ech AB, Uppsala, Sweden) and sc eened by Wes e n blo ing wi h h ee po cine se a (a hype immune se um om a pig accina ed h ee imes wi h Salmopo c, a pool o ield se a om pigs posi i e by he Salmo ype PigSc een ELISA, and a pool o se a om pigs expe imen ally in- ec ed wi h S.en e ica se o a De by), each used a a 1:200 dilu ion. Blo s we e de eloped using an alkaline phospha ase-conjuga ed goa an i-swine immuno- globulin G (Diano a, Hambu g, Ge many) and BCIP (5-b omo-4-chlo o-3-in- dolylphospha e) and ni oblue e azolium as a ch omogenic subs a e. P o ein spo s eac ing s ongly wi h all h ee se a we e selec ed o iden i ica ion ia Q-TOF MS/MS. Iden i ica ion o immunogenic p o eins. Coomassie blue-s ained spo s co e- sponding o immunogenic p o eins we e cu om a 2D PAGE gel, ypsina ed, and elu ed om he gel by using a me hod sligh ly modi ied om ha desc ibed p e iously by Wilm e al. (50). B ie ly, he gel pieces we e dehyd a ed wi h 100 ␮l ace oni ile, ehyd a ed wi h 30 ␮l ehyd a ion bu e (100 mM NH 4 HCO 3 con aining 10 mM di hio h ei ol), and hen ea ed wi h 100 mM iodoace amide in 30 ␮l 100 mM NH 4 HCO 3 . Dehyd a ion and ehyd a ion we e epea ed, and he dehyd a ed gel pieces we e hen ehyd a ed wi h 3 o 15 ␮l ehyd a ion bu e con aining 20 ng/␮l ypsin (sequencing g ade) o 12 o 16 h a 37°C. Pep ides we e ex ac ed using 50 mM NH 4 HCO 3 ollowed by a solu ion con aining 50% ( ol/ ol) ace oni ile and 5% ( ol/ ol) o mic acid. A e e apo a ion o all liquid in a acuum cen i uge, pep ides we e esuspended in a solu ion con aining 50% ( ol/ ol) ace oni ile and 0.1% ( ol/ ol) o mic acid. Pep ide sequences we e de e mined om MS/MS agmen a ion da a eco ded on an ESI Q-TOF mass spec ome e (Q-To Ul ima; Wa e s, Mil o d, MA). P o eins we e iden i ied by using he p og am P o einLynx Globals Se e ( e sion 2.1; Wa e s) and by sea ching he Na ional Cen e o Bio echnology In o ma ion (NCBI) comple e da abase ( p:// p.ncbi.nlm.nih.go ./BLAST/db/FASTA/). Iden i ica ion and syn hesis o immunogenic epi opes. Epi ope mapping was pe o med by pep ide spo a ay analysis using o e lapping 15-me pep ides ini ia ing a e e y hi d amino acid (12, 13). The pep ide-coa ed memb ane was we ed wi h e hanol o enhance he sol a ion o hyd ophobic pep ide spo s and hen washed h ee imes wi h T is-bu e ed saline (TBS) (10 mM T is [pH 7.0], 154 mM NaCl [pH 7.0]) o 10 min and inally incuba ed o e nigh a 4°C in 10 ml o memb ane blocking solu ion (pH 7.0) (80% [ ol/ ol] TBS–0.05% Tween 20 [T-TBS; pH 8.0], 20% [ ol/ ol] casein-based blocking bu e concen a e [Geno- sys Bio echnology, Camb idge, England], 5% [w / ol] suc ose). A e washing wi h 10 ml T-TBS (pH 7.0), he memb ane was incuba ed wi h po cine se um aised agains he S. en e ica se o a Typhimu ium accine s ain Salmopo c o 2 h (dilu ed 1:200 in memb ane blocking solu ion). The blo was de eloped as desc ibed abo e. The ch omogenic eac ion was s opped by washing blo s wice wi h phospha e-bu e ed saline. The memb ane was s ipped using 20 ml o N,N-dime hyl o mamide wice o 10 min o dissol e he blue colo o spo signals, ollowed by washing h ee imes wi h wa e , s ipping mix A (8 M u ea, 1% sodium dodecyl sul a e, and 0.5% me cap oe hanol in phospha e-bu e ed saline [pH 7.0]), s ipping mix B (10% ace ic acid, 50% e hanol, 40% wa e ), and e hanol, espec i ely. A e wa ds, blo ing was epea ed wi h po cine nega i e se um (a pool o Salmo ype PigSc een ELISA-nega i e se a om pigs om a Salmonella- ee he d) in o de o exclude unspeci ic epi opes. Pu a i e immunogenic epi opes (noname s) we e syn hesized wi h an amino- e minal bio inyla ion linked by a 2-aminohexanoic acid linke and pu i ied by high-pe o mance liquid ch oma og aphy (Pep ide Special y Labo a o ies GmbH, Heidelbe g, Ge many). Lyophilized pep ides we e esuspended in dis- illed wa e o ob ain s ock solu ions o 5 and 10 mg/ml, espec i ely. Pu a i e immunogenic epi opes and su ace-exposed domains we e p edic ed using he algo i hms “An igenic” and “B2TMR-HMM” (18, 20, 23, 29), espec i ely. Cons uc ion o an isogenic S. en e ica se o a Typhimu ium Salmopo c⌬ompD s ain. A unca ed ompD gene wi h an in- ame dele ion was cons uc ed using a class IIs es ic ion endonuclease app oach (38). Two PCR p oduc s o 983 bp and 792 bp we e gene a ed using p ime s oDWST5_ou a/oOMPDKO1 and oOMPDKO2/oDWST5_ou b, espec i ely (Table 1). Bo h agmen s we e e- s ic ed wi h BsmBI, liga ed, eampli ied, and cloned in o pTOPO2.1 (In i o- gen, Heidelbe g, Ge many). The inse was con i med by nucleo ide sequence analysis, emo ed by SacI es ic ion, and liga ed in o he mu agenesis ec o pROKB2 es ic ed wi h SacI, esul ing in plasmid pSOM14666. The plasmid was ans o med in o he dono s ain Esche ichia coli ␤2155 and mobilized in o S. en e ica se o a Typhimu ium Salmopo c by il e ma ing. The dono was g own on LB aga pla es supplemen ed wi h kanamycin (50 ␮g/ml) and diaminopimelic acid (10 mM) a 37°C o 16 h, and he ecipien was g own on Columbia sheep VOL. 75, 2007 S. ENTERICA SEROVAR TYPHIMURIUM DIVA VACCINE 2477 on Janua y 20, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://iai.asm.o g/Downloaded om blood aga (Oxoid GmbH, Wesel, Ge many) a 37°C o 44 h. Bac e ia we e ha es ed wi h s e ile co on swabs and esuspended in TNM bu e (1 mM T is-HCl [pH 7.2], 100 mM NaCl, 10 mM MgSO 4 ) oanOD 600 o 1. Fil e ma ing was pe o med as desc ibed p e iously (28); he conjuga ion mix u e was pla ed on o LB aga pla es supplemen ed wi h kanamycin (40 ␮g/ml) and incu- ba ed a 37°C o e nigh . Colonies we e subcul u ed and sc eened ia PCR wi h ompD-speci ic p ime s oOMPD1 and oOMPD2. Colonies con aining bo h he wild ype and he unca ed gene we e conside ed o be po en ial genomic coin eg a es and we e used o coun e selec ion. Coun e selec ion. A single colony o S. en e ica se o a Typhimu ium Salmo- po c coin eg a e was inocula ed o e nigh in LB b o h. Sal - ee LB b o h (2.5 ml) con aining 10% suc ose was inocula ed wi h 2.5 ␮l o he cul u e g own o e nigh and kep a 37°C in a shaking incuba o (200 pm) o 4 h. In o de o imp o e coun e selec ion e icacy, he cul u e was subsequen ly s o ed a 4°C o 72 h, and 50-␮l aliquo s we e pla ed on o LB aga . Single colonies we e eplica pla ed on o LB aga wi h (40 ␮g/ml) and wi hou kanamycin. The geno ype o kanamycin-sensi i e colonies was de e mined by PCR wi h p ime s oOMPD1 and oOMPD2 and e i ied by Sou he n blo analysis, nucleo ide sequencing analysis, and pulsed- ield gel elec opho esis. Vi ulence s udy in BALB/c mice. Vi ulence o S. en e ica se o a Typhimu ium Salmopo c⌬ompD was assessed in an in ec ion model by de e mining he 50% le hal dose (LD 50 ) in BALB/c mice in compa ison o he pa en s ain Salmo- po c. The animals, 32 emale 17- o 20-g BALB/c mice, we e in ec ed pai wise wi h 1 ⫻10 2 o1⫻10 8 CFU o ei he he pa en o he mu an s ain and TABLE 1. Bac e ial s ains, plasmids, and p ime s used in his s udy S ain, plasmid, o p ime Cha ac e is ic(s) Sou ce o e e ence S ains E. coli ␤2155 h B1004 p o hi s A hsdS lacZ⌬M15 (F⬘lacZ⌬M15 laqI q aD36 p oA ⫹ p oB ⫹ )⌬dapA::e m (E m ) ecA::RPA-2- e (Tc )::Mu-Km (Km )␭pi 8 E. coli TOP10 F ⫺ mc A ⌬(m -hsdRMS-mc BC)␾80lacZ⌬M15 ⌬lacX74 ec A1 deoR a aD139 ⌬(a a leu)7697 galU galK psL (S )endA1 nupG TOPO TA cloning; In i ogen S. en e ica se o a Typhimu ium 421/125 S. en e ica subsp. en e ica se o a Typhimu ium li e accine s ain p esen in he licensed accine Salmopo c IDT, Dessau-To nau, Ge many S. en e ica se o a Typhimu ium 421/125⌬ompD Unma ked ompD-nega i e knockou mu an o S. en e ica se o a Typhimu ium 421/125 This wo k S. en e ica se o a Typhimu ium DT104 27/96 S.en e ica se o a Typhimu ium ield isola e ca ying phage ype DT104 wi h Amp Clin E y Pen Te 44 Plasmids pROCKB2 8.9-kb ansconjuga ion ec o based on pROKB1 wi h o iR6K and mobRP4,Ap Km , polycloning si e, sacB This wo k pSOM14666 T ansconjuga ion ec o pROCKB2 ca ying a unca ed e sion o ompD wi h o iR6K,mobRP4,Ap Km , polycloning si e, sacB This wo k pTOPO 2.1 Topoisome ase I-“enhanced” E. coli cloning ec o ca ying ampicillin and kanamycin esis ance de e minan s as well as a lacZ gene o blue-whi e selec ion TOPO TA cloning; In i ogen pSOM810 Vec o pTOPO 2.1 ca ying PCR p oduc o p ime s pOMPDKOMP1 and pOMPDKOMP2, s a ing 516 bp ups eam o he ompD s a codon and ending 31 bp downs eam o he s op codon, o complemen a ion o S. en e ica se o a Typhimu ium ⌬ompD This wo k P ime s oDWST5ou a 5⬘-TGC TCTAGA CCC GGA GAA ATT ATC AGC AA-3⬘( o wa d p ime con aining an XbaI es ic ion si e on he 5⬘end si ua ed 974 bp ups eam o he ompD s a codon) This wo k oDWST5ou b 5⬘-GCC GAGCTC AGA GAT TGC CAG AGC GTC AT-3⬘( e e se p ime con aining a SacI es ic ion si e on he 5⬘end si ua ed 753 bp downs eam o he ompD open eading ame) This wo k oOMPDKO1 5⬘-CGTCTCGAAGTTTCATTTTAATAATCCTTAT-3⬘( e e se p ime wi h BsmBI es ic ion ecogni ion si e on 5⬘end si ua ed 9 bp down- s eam o he ompD s a codon; e e se p ime o oDWST5_ou a o ampli ica ion o he ups eam DNA agmen used o cons uc a un- ca ed ompD) This wo k oOMPDKO2 5⬘-CGTCTCGACTTCTGAACTACCAGTTCTAATT-3⬘( o wa d p ime wi h BsmBI es ic ion ecogni ion si e on 5⬘end si ua ed 2 bp down- s eam o he ompD s op codon; o wa d p ime o oDWST5_ou b o ampli ica ion o he downs eam DNA- agmen used o cons uc a unca ed ompD) This wo k oOMPD1 5⬘-CCATACCAGGATTGCGCTG-3⬘( o wa d p ime si ua ed 393 bp ups eam o he ompD s a codon) This wo k oOMPD2 5⬘-CGGTAAGCCGAAACCACAG-3⬘( e e se p ime si ua ed 317 bp downs eam o he ompD open eading ame) This wo k oOMPDKOMP1 5⬘-GGCGGGCCGATATTGATATT-3⬘( o wa d p ime o complemen a- ion o S. en e ica se o a Typhimu ium ⌬ompD si ua ed 516 bp up- s eam o he ompD s a codon; he 516-bp nucleo ide sequence was also analyzed o p omo e s using he Web-based p og am BPROM [www.so be y.com] o ensu e ha he p omo e o he ompD gene was included) This wo k oOMPDKOMP2 5⬘-GGACTGGCTTTGTATTCAGAC-3⬘( e e se p ime o complemen- a ion o S. en e ica se o a Typhimu ium ⌬ompD si ua ed 31 bp down- s eam o he ompD s op codon) This wo k 2478 SELKE ET AL. INFECT.IMMUN. on Janua y 20, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://iai.asm.o g/Downloaded om obse ed o 19 days pos in ec ion. Spleens and li e s o animals dying du ing he ime o obse a ion we e cul u ed o possible eisola ion o he espec i e s ain; isola ed Salmonella colonies we e iden i ied using he IDT Salmonella diagnos ic ki and a PCR wi h ompD-speci ic p ime s, acili a ing he di e en ia ion o Salmopo c⌬ompD and Salmopo c. The LD 50 was calcula ed using P obi analysis so wa e (SPSS Inc., Chicago, IL). Vaccina ion ial in pigs. The Salmopo c⌬ompD s ain cons uc ed in his s udy was used as an o al accine in a accina ion s udy in pigs (Ge man Land- ace) and compa ed o he pa en s ain (Salmopo c) and a placebo g oup. The ial was pe o med essen ially as desc ibed p e iously (41); an S.en e ica se o a Typhimu ium DT104 clinical isola e (s ain 27/96) was used as a challenge s ain. The animal expe imen included h ee g oups o six pigs each, 4 weeks o age, accina ed o ally wi h placebo, S.en e ica se o a Typhimu ium Salmopo c⌬ompD (6 ⫻10 8 CFU/ml pe accina ion), o S.en e ica se o a Typhimu ium Salmo- po c (acco ding o manu ac u e ’s ins uc ions), espec i ely. Th ee addi ional pigs we e accina ed wi h S.en e ica se o a Typhimu ium Salmopo c⌬ompD o in es iga e he de elopmen o an ibody i e s un il 3 weeks a e in ec ion. Two ounds o accina ion we e pe o med 3 weeks apa . Blood samples we e aken p io o he i s (day 0) and second (day 21) immuniza ions, 1 week be o e challenge, and a nec opsy. Fo he h ee pigs kep un il 3 weeks a e in ec ion, addi ional blood samples we e aken 1, 2, and 3 weeks pos challenge (see Table S4 in he supplemen al ma e ial). Animals we e ca ed o in acco dance wi h he p inciples ou lined in he Eu opean Con en ion o he P o ec ion o Ve eb a e Animals Used o Expe imen al and O he Scien i ic Pu poses (Eu opean T ea y Se ies no. 123 [h p://con en ions.coe.in / ea y/EN/Menup incipal.h m]; pe mi no. 06/1066). In ec ion o pigs. Fo o al in ec ion on day 43, a 50-ml cul u e (LB medium) was inocula ed wi h 5 ml o a liquid cul u e o S.en e ica se o a Typhimu ium DT104 s ain 27/96 (LB medium) g own o e nigh and was g own wi h shaking a 37°C o an OD 600 o 0.9. The cul u e was placed on ice o 10 min and kep on ice un il use o a maximum o 2 h. Pigle s we e in ec ed wice wi hin 2 hou s, and inoculum doses we e de e mined immedia ely a e in ec ion ia se ial di- lu ion and subsequen pla ing on o Rambach aga pla es supplemen ed wi h ampicillin (100 ␮g/ml). In ec ions we e ca ied ou using a ben -knob cannula and sy inge o adminis e he app op ia e dose o S.en e ica se o a Typhi- mu ium DT 104 s ain 27/96 o he pigle s 3 weeks a e he second immuniza ion ia he o al ou e (5 ⫻10 8 CFU/ml pe ound, esul ing in a o al dose o 1 ⫻10 9 CFU/ml). Su eillance o animals. Body empe a u e, eeding beha io , and clinical symp oms we e eco ded a leas daily o each indi idual pig o as needed. A clinical sco ing sys em was employed o assess he clinical condi ion o each indi idual animal as ollows. A sco e o 1 each was gi en o he occu ence o e e ( ec al empe a u e ⬎40°C), lack o appe i e, and dia hea/ omi us/le h- a gy, esul ing in a minimum clinical sco e o 0 and a maximum sco e o 4 pe day; he added daily clinical sco es o days 1 o 7 we e designa ed he o al clinical sco e. S a is ical analysis o he o al clinical sco e was pe o med using he Wilcoxon es . Animals we e bled on days 0, 36, 50, 57, and 65 du ing he cou se o he expe imen . Bac e iological examina ion o o gan samples. In o de o de e mine he p o ec i e e icacy o he accine wi h espec o coloniza ion, o gan samples (ileocecal lymph nodes, ileum [app oxima ely 10 cm c anial o he ileocecal al e], and cecum [apex]) we e aken a pos mo em examina ion. In es inal o gan samples we e washed, and 0.1 g o mucosa was emo ed by sc aping wi h a scalpel, aken up o 1 ml wi h saline, and homogenized using a bead bea e (speed o 5.0, 40 s/ ound; The moSa an ) wi h h ee glass beads (diame e , 3 mm). Ten old se ial dilu ions we e pla ed on o Rambach aga con aining 100 ␮g/ml o ampicillin in o de o supp ess he g ow h o o he En e obac e iaceae and incuba ed o e nigh a 37°C. he Salmonella colonies g own we e coun ed, he numbe o CFU/ml was calcula ed, and indi idual colonies we e con i med o be exempla y by an ibio ic esis ance yping and S.en e ica se o a Typhimu ium DT104-speci ic mul iplex PCR (32). ELISAs. Fo he OmpD-speci ic pep ide-based ELISA, Nunc Immobilize S ep a idin F96 mic o i e pla es we e incuba ed o 1 h wi h 100 ␮l o OmpD- de i ed syn he ic, bio inyla ed pep ide (posi ions F 100 o Y 108 o he OmpD p o ein; 25 ␮g/ml) as a solid-phase an igen. Po cine se a we e p eabso bed wi h a whole-cell lysa e o S.en e ica se o a Typhimu ium Salmopo c⌬ompD o 1h in o de o emo e possible c oss- eac i e an ibodies di ec ed agains simila epi opes o o he po ins. Se ial wo old dilu ions o he p eabso bed se a (s a - ing wi h a dilu ion o 1:10) we e added and incuba ed o 1ha oom empe - a u e. The ELISA was de eloped using goa an i-pig pe oxidase conjuga e (Diano a) and 2,2-azino-di-[3-e hylbenzi hiazoline sul ona e] (ABTS; Roche Di- agnos ics, Mannheim, Ge many) as a subs a e. To de e mine he i e o an i- Salmonella LPS an ibodies, he comme cial Salmo ype PigSc een ELISA (Labo Diagnos ik GmbH, Leipzig, Ge many) was ca ied ou acco ding o he manu- ac u e ’s ins uc ions. Nucleo ide sequence accession numbe . The nucleo ide sequence o plasmid pRouB2 has been submi ed o GenBank unde accession numbe AM180348. RESULTS Iden i ica ion o a sui able ma ke p o ein. To iden i y su - ace-exposed, immunogenic p o eins o he licensed li e ac- cine Salmopo c, exponen ially g owing bac e ia we e ha es ed and ea ed wi h sodium deoxychola e o en ich ou e mem- b ane p o eins in he supe na an ia de e gen wash. By sep- a a ing he en iched ou e memb ane p o eins by 2D-PAGE and sc eening hei immunogenici y by Wes e n blo analyses (see Fig. S1 in he supplemen al ma e ial), we iden i ied i e highly immunogenic p o eins (see Table S1 in he supplemen- al ma e ial). They eac ed s ongly wi h a pool o se a om animals accina ed h ee imes wi h Salmopo c as well as wi h pools o se a om animals in ec ed wi h S.en e ica se o a Typhimu ium o S.en e ica se o a De by, espec i ely. The p o eins co esponding o he immuno eac i e spo s we e cu om a Coomassie blue-s ained 2D-PAGE gel and iden i ied a e ypsin diges ion using Q-TOF MS/MS. Fou p o eins could be iden i ied unambiguously as he majo po ins OmpC and OmpD, a homolog o an ou e memb ane p o ein o Acine obac e spp., and elonga ion ac o Tu. Cha ac e iza ion o OmpD as a pu a i e nega i e ma ke . In silico analyses o he candida e p o eins esul ed in he selec- ion o ou e memb ane po in D (OmpD) as a pu a i e nega- i e ma ke . By pe o ming a mapping o con inuous an igenic epi opes o he OmpD p o ein by pep ide spo a ay analysis (see Fig. S2 in he supplemen al ma e ial) wi h o e lapping 15-me s, we iden i ied ou highly immunogenic epi opes h oughou he p o ein. These ma ched only pa ially wi h pu- a i e immunogenic epi opes and su ace-exposed domains p edic ed using he algo i hms “An igenic” (see Table S2 in he supplemen al ma e ial) and “B2TMR-HMM” (see Table S3 in he supplemen al ma e ial), espec i ely. All ou epi opes we e syn hesized as bio inyla ed pep ides and es ed as solid-phase an igens o hei applicabili y in an OmpD- speci ic ELISA, wi h pep ide 2 acili a ing he bes disc imina- ion be ween OmpD an ibody-posi i e and -nega i e se a (Fig. 1). Gene a ion o an ompD-de icien mu an s ain. An isogenic S.en e ica se o a Typhimu ium Salmopo c⌬ompD mu an was cons uc ed by allelic exchange using he suicide plasmid pSOM14666 (con aining an in- ame dele ion o ompD) (see Fig. S3 in he supplemen al ma e ial) upon conjuga ion wi h E. coli ␤2155 as he dono s ain. Plasmid coin eg a es we e se- lec ed on kanamycin, con i med by PCR, and esol ed by su- c ose coun e selec ion. Suc ose- esis an and kanamycin-sen- si i e colonies we e con i med by PCR, nucleo ide sequencing, and Sou he n blo analysis as well as pulsed- ield gel elec o- pho esis (see Fig. S4 in he supplemen al ma e ial). The phe- no ype was con i med by one-dimensional PAGE (Fig. 2) and subsequen Q-TOF MS/MS. The mu an gene a ed is ee o o eign DNA and was designa ed S.en e ica se o a Typhi- mu ium Salmopo c⌬ompD. Vi ulence in BALB/c mice. In o de o ensu e an unchanged i ulence o he mu an s ain, we challenged 16 g oups o wo BALB/c mice each wi h eigh di e en doses o Salmopo c and VOL. 75, 2007 S. ENTERICA SEROVAR TYPHIMURIUM DIVA VACCINE 2479 on Janua y 20, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://iai.asm.o g/Downloaded om Salmopo c⌬ompD and de e mined he LD 50 o be app oxi- ma ely 1.3 ⫻10 8 CFU o bo h s ains (see Table S4 in he supplemen al ma e ial). These esul s demons a ed ha he lack o ompD does no addi ionally a enua e S.en e ica se o- a Typhimu ium Salmopo c o BALB/c mice. P o ec i e e icacy in pigs. Fou clinical symp oms ( e e [ⱖ40.0°C], le ha gy, educed ood up ake, and en e i is [ om- i us/dia hea]) we e added o esul in a clinical sco e wi h a maximum o 4 pe animal and day. A signi ican di e ence (P⫽ 0.046; Wilcoxon es ) be ween ei he o he accina ed g oups and he placebo g oup was obse ed (Fig. 3). All animals in he placebo g oup de eloped e e 2 days pos in ec ion and showed educed eed up ake, whe eas none o he accina ed animals de eloped e e , and only wo accina ed animals showed hesi an eed up ake. No signi ican di e ence be ween he g oups accina ed wi h S.en e ica se o a Typhimu ium Salmopo c and S.en e ica se o a Typhimu ium Salmopo c- ⌬ompD was obse ed. One week pos in ec ion, animals we e nec opsied. All pigs showed eac i e hype emia in he ileum and cecum, wi h some o he placebo- accina ed animals showing highly eac i e hy- pe emia in he cecal mucosa. Salmonella coloniza ion was de- e mined o h ee de ined loca ions (ileal and cecal mucosa and ileocecal lymph node). Fo he cecum (P⫽0.042; F ied- mann es ) and ileocecal lymph nodes (P⫽0.007; F iedmann es ), accina ion wi h ei he accine signi ican ly educed col- oniza ion (Table 2). Howe e , he educ ion o coloniza ion was abou 10- old lowe upon accina ion wi h S.en e ica se - o a Typhimu ium Salmopo c⌬ompD han upon accina ion wi h he S.en e ica se o a Typhimu ium Salmopo c pa en s ain (Table 2). Ma ke p ope ies o he OmpD p o ein. In addi ion o he p o ec i e e icacy, he unc ionali y o he nega i e ma ke OmpD was in es iga ed in he accina ion s udy. In o de o unc ion as a se ologically de ec able ma ke , an ibodies di- FIG. 1. Disc imina o y e icacy o OmpD-de i ed immunogenic epi opes. Bio inyla ed epi opes 1 o 4 we e used as solid-phase an i- gens on s ep a idin-coa ed ELISA pla es and incuba ed wi h in e nal posi i e and nega i e con ol se a, espec i ely, ob ained om animals in he accina ion ial. The ba s show he means and s anda d de i- a ions o h ee independen ELISA expe imen s. OD%, ela i e op i- cal densi y o he nega i e con ol se um compa ed o ha o he posi i e con ol se um (de ined as 100 OD%) o each coa ing pep ide. FIG. 2. Cha ac e iza ion o S.en e ica se o a Typhimu ium Salmopo c⌬ompD. Pheno ypes we e cha ac e ized using memb ane p epa a ions o Salmopo c (lane 1), Salmopo c⌬ompD (lane 2), and Salmopo c⌬ompD complemen ed in ans wi h plasmid pSOM810 (lane 3). P o ein bands A o C we e sequenced using Q-TOF MS/MS and iden i ied as being OmpC (A), OmpD (B), and OmpF (C), e- spec i ely. LMW, low molecula weigh ma ke (Ame sham Pha mcia Bio ech AB, Uppsala, Sweden). FIG. 3. Clinical sco es o pigs in he accina ion and challenge ial. The o al sco es o indi idual animals o days 1 o 7 pos in ec ion and he a i hme ic means (ho izon al ba s) calcula ed o g oups 1 o 3 (six animals each) a e gi en. The as e isks indica e s a is ical signi icance (Wilcoxon es ), and he P alues a e gi en. TABLE 2. Reisola ion o he challenge s ain om ileocecal lymph nodes, cecum, and ileum Tissue Reisola ion o challenge s ain (CFU/g issue) a Non accina ed Salmopo c⌬ompD Salmopo c Ileocecal lymph nodes 1.21 ⫾10 4 *5.13 ⫾10 2 *6.67 ⫾10 1 * Cecum 6.79 ⫾10 4 *2.67 ⫾10 2 *1.67 ⫾10 1 * Ileum 7.30 ⫾10 4 1.67 ⫾10 3 6.67 ⫾10 2 a An ⴱindica es ha he alues we e signi ican ly lowe (P⬍0.05; F iedmann es ) han hose in he non accina ed con ol g oup. 2480 SELKE ET AL. INFECT.IMMUN. on Janua y 20, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://iai.asm.o g/Downloaded om ec ed agains he OmpD p o ein ha e o be de eloped by he hos upon in ec ion wi h S.en e ica se o a Typhimu ium o accina ion wi h a comme cial S.en e ica se o a Typhi- mu ium accine bu no upon accina ion wi h he ma ke accine s ain S.en e ica se o a Typhimu ium Salmopo c- ⌬ompD. Thus, a se ological di e en ia ion o in ec ed and ac- cina ed pigs is made possible (DIVA p inciple). Salmonella an ibody i e s as assessed in he Salmo ype Pig- Sc een ELISA s eadily ose du ing he s udy in bo h accina ed g oups, whe eas i e s in he placebo g oup s ayed nega i e (see Fig. S5 in he supplemen al ma e ial). An ibody i e s in he OmpD-speci ic pep ide-based ELISA de eloped in he S. en e ica se o a Typhimu ium Salmopo c- accina ed g oup bu emained nega i e be o e challenge in he S.en e ica se o a Typhimu ium Salmopo c⌬ompD- and placebo- accina ed g oups (Fig. 4) (day 0 and day 36). Sho ly a e challenge, OmpD-speci ic se ocon e sion was obse ed in he S.en e ica se o a Typhimu ium Salmopo c⌬ompD- and placebo- acci- na ed g oups (Fig. 4) (day 50). Th ee animals accina ed wi h S.en e ica se o a Typhimu ium Salmopo c ⌬ompD be o e in ec ion we e moni o ed se ologically up o 3 weeks pos chal- lenge. OmpD-speci ic an ibody i e s emained posi i e un il he end o he expe imen (Fig. 4) (days 57 and 65). DISCUSSION In his epo , we desc ibe he iden i ica ion o a DIVA ma ke , i s in oduc ion in o he con en ionally a enua ed li e accine s ain Salmopo c, and he de elopmen o a disc imi- na o y ELISA sys em. Salmopo c was chosen o his wo k because i has been shown o educe bo h shedding and colo- niza ion o he po cine in es inal ac (41). The app oach de eloped is s aigh o wa d and migh be gene ally applicable o he cons uc ion o li e bac e ial DIVA accines. The adap a ion o a widely used allelic exchange s a egy wi h sacB as he coun e selec able ma ke allowed he con- s uc ion o a Salmonella accine s ain ca ying an in- ame dele ion o he ompD gene no con aining o eign DNA. Al- hough his me hod is mo e labo ious han he commonly used PCR-based allelic exchange desc ibed p e iously by Da senko and Wanne (6), his app oach migh be ad an ageous o accine de elopmen and subsequen licensing. Thus, he e- sul ing s ain is indis inguishable om a spon aneously occu - ing dele ion mu an and he e o e is no conside ed a gene ically modi ied o ganism acco ding o Eu opean egula ions on gene ic enginee ing (di ec i e [EC] 2001/18; h p://eu opa.eu/eu -lex/p i /en/oj/da /2001/l_106/l_10620010417en00010038.pd ). The p o ein OmpD, chosen as selec able ma ke , is one o he mos abundan p o eins in he ou e memb ane o Salmo- nella, ep esen ing abou hal o he 1 ⫻10 5 o2⫻10 5 po in molecules pe cell unde a o able g ow h condi ions (27, 36). I is p esen in all S.en e ica se o a s wi h he excep ion o S. en e ica se o a Typhi (35, 37). In con as o he majo po in OmpC, howe e , OmpD is no ound in o he g am-nega i e bac e ia (40). The p esence o OmpD and o he po ins in he de e gen wash ac ion is likely due o he p esence o mem- b ane esicles (blebs) commonly o med by g am-nega i e bac- e ia (31, 52). On he one hand, i s high abundance makes OmpD a highly a ac i e choice as a nega i e ma ke . Thus, se ocon e sion is likely o occu upon in ec ion o pigs wi h any Salmonella se o a (excep S.en e ica se o a Typhi), he eby ac i a ing he DIVA unc ion. This b oad-spec um DIVA unc ion is manda o y o a po cine Salmonella accine, as legisla ion is no limi ed o ce ain se o a s. The e o e, con- en ional ma ke s ains, such as ough mu an s (39, 49), can- no be used. On he o he hand, OmpD is homologous o o he po ins common among En e obac e iaceae. The e o e, in o de o ob ain a su icien ly disc imina o y ELISA e icacy, no he en i e OmpD p o ein bu an OmpD-speci ic pep ide selec ed by pep ide spo a ay analyses had o be used as a solid-phase an igen. In addi ion o he mo e di icul se up o a disc imina o y se ological es , he use o a majo immunogenic p o ein as a DIVA an igen has wo o he possible d awbacks. Thus, he dele ion migh (i) cause an addi ional a enua ion o he mu- an s ain and (ii) diminish p o ec i e e icacy. Fo he dele- ion o OmpD om S.en e ica se o a Typhimu ium, epo s wi h espec o a enua ion a e con o e sial (10, 25). Bo h s udies, howe e , ha e been ca ied ou using an ompD mu an ob ained by Tn10-based mu agenesis (10, 25). In o de o en- su e unchanged i ulence o he S.en e ica se o a Typhi- mu ium Salmopo c⌬ompD s ain, we ca ied ou an LD 50 de- e mina ion in BALB/c mice. This expe imen unambiguously showed equal i ulence o bo h s ains. The accina ion s udy pe o med demons a ed ha pigs accina ed wi h ei he accine we e p o ec ed equally well om clinical symp oms, he eby con i ming he esul s om he mouse expe imen . On he o he hand, he DIVA accine showed a educed e icacy in compa ison o he Salmopo c pa en s ain wi h espec o educing o gan coloniza ion. Thus, he numbe o S.en e ica se o a Typhimu ium DT104 cells o be isola ed om ileocecal lymph node, cecum, and FIG. 4. Se ological esponses o pigs in a disc imina o y ELISA upon immuniza ion and challenge. Animals we e immunized on days 1 and 21 and challenged on day 43. Blood was aken on days 0, 36, and 50 (six animals pe g oup) and on days 57 and 65 ( h ee animals). An i-OmpD an ibody i e s we e de e mined using an ELISA wi h OmpD-de i ed pep ide 2 as a solid-phase an igen. The ho izon al line ma ked wi h “⫹” (OD% ⫽100) indica es he posi ion o he in e nal posi i e con ol consis ing o he pooled se a om day 50. OD%, ela i e op ical densi y o he ex se a compa ed o ha o he in e nal posi i e con ol se um (de ined as 100 OD%). VOL. 75, 2007 S. ENTERICA SEROVAR TYPHIMURIUM DIVA VACCINE 2481 on Janua y 20, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://iai.asm.o g/Downloaded om ileum was 10- old highe in pigs immunized wi h Salmopo c- ⌬ompD han in hose immunized wi h he Salmopo c pa en s ain. These esul s imply ha a dele ion o he OmpD p o- ein, al hough o no consequence wi h espec o causing sep- icemia in mice upon in ape i oneal applica ion, migh educe coloniza ion and su i al upon o al applica ion in he po cine gas oin es inal ac . Howe e , he DIVA accine s ain s ill educes S.en e ica se o a Typhimu ium eisola ion a es sig- ni ican ly (10- o 100- old). As an in ec ion occu ing a e accina ion is no masked, he S.en e ica se o a Typhimu ium Salmopo c⌬ompD ac- cine s ain, despi e i s educed e icacy in educing coloniza- ion, migh be a aluable ool in se osu eillance-based Salmo- nella con ol p og ams aimed a educing he isk o human in ec ion. The ac ha S.en e ica se o a Typhimu ium Salmopo c⌬ompD does no con ain o eign DNA and ha i s pa en s ain, S.en e ica se o a Typhimu ium Salmopo c, has been used ex ensi ely in he ield should acili a e he ealiza- ion o u u e ield s udies and subsequen licensing p oce- du es. 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