AU2003204710B2 - Attenuated microorganisms for the treatment of infection - Google Patents

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AU ST RAUA Patents A.19 ,,OMIPLETE SPEQIFIOATION DIVISIONAL PATENT A PPUiCANT: Invention Tile: Microscience Limited Attenuated Mircorganisnis tor the Triatrneat of tnfection The -fowng ci statement is a 14~ desc -ptinJn ot this nva et o n :ciudg the, bestM mO o p, n OWforirq ill knao ir- moe, lWNO (MW659261 PCT"(QBOU/0J I: ATTNUA ,-DM NCO- kOAN r' FOR THE TRE-ATMENT OF WNFECTDQN ofr the ofrventir wa ifeio Tis rWTentio relat fio a tenuted m co rgass tate hip.n' be uezbi Vacies vatofe omosporme th evflt s ar-ctreamare ofbteriior iale mnton and of longer duration then lThose produced by non repfieatnqg rnmwnapefs. onse ana~or ~r tismay L~ V4 b Stkerualleo strainc estlish limizeo infecwtnm&,n tbe hd%* and. ime Mi the earhy stages o-f natural infectirln :in adtn.unlik*e xifled prear~iof I~ ve v&arfmmrm b ostc ctn eimeitcrspne ht aybe cornettd with lheir abiliytrpitaep r eetn cal. ucph 21 There has. been, a long history ofl DhS ut- iv li ve trwated Sloel vaccines as sate mnd effective vaccines for the prev, ento fsrnnelssi and humans. lnceed, the live atenuated orattphi vacc inet T,~s{ivt manlufactured by' th~e Swfss serurn vaccine institute, ha provevd to be avery, suoesfivacci-ne for the prevention, of typhoifd fever and has been licanz ed mn many courntries inokucing ie US and Europe.

Ho)weve. the itteiwu-abon 0,Fhi sa-i wa,,s achievetd Using cherniot: muaenests tek-hliqjis end the basks of attenuation, of *me strait is no tly anderstoo a-eecatrse of this, the vcit not ideal in terms of the nuntttfl oi ooseS, ~currsntiy four) and the number of lieorcenisms that have to be given atechds Modem rnoecuiar boiogy techniques, uouvld with the increasing iknowledge of Sannih amgflss a ied to the identificationc of se eral genes that are essential for the in Vivo growth and surviva.l of the orqatnm This has pruvtoed new oeetrets lor nemaion eadin to thie concept that future vacbim strains, canb tatfonaiy' aftenuate so b intoducing defined non-revertmnpuaoslrosett Q genes known to be involv-ed in vfule&ce. This wifll facihtate the di:svelopml of imnrovO vcines. pmrticularfy in terro, of themunogenici^y and therefore kthe number of doses that have to be oiven.

Aftttough many attenuateid strzains ocSalinei are now imoonwrk. te Zw have qafidas,-, tcivcie iaa f-or us:e i humns. This may be.r. 'npan Wi) 04i6&t *t to tMe nzeed to badanCe the-. itoc muoepivo hevwn ihh orzsi bdty ofth it ie clear, that the teection of prpit tatqe tsz for attenuationl wk)ct w rzsit~n stbe vccne-Ci~dt~t, nt trImhfotwam and Cannot ativbe a ppsot va7-ccine, and there i much research:- beiantq carhc oWt identi sabl .Ktr~s Qxampie. mwny a tae sri el tas v; um 'RCO1 nd auICse 1Lao to vactfle~abascesses sin thle patipnt it s tereoredesirabie wo devdlop a vaccie having a hl creu lrrnnoenciy I:h fr possibilty o'hte mirognsmsr0 evrigt an recie torm and wnichl exhibits, a gOod safet'y profle with brahezd sie ffects, h l x nto 5rsn"rnt r r r~sc 1tefn~qht w pcfcxrnan mrsnatonsmt ducd ~ro Sainoe~amicroupan rm can proc,,uce avciehvn hnh egee itrmiungenciy ane o if f the iimorlnism revertota reainve torn The! reu.n vacine strifons, exhibit a dO i f~o po~e stenoi two (Sp12,: the so~c in~:~uotrp mutltinr. ixe. a. niiutatixi to disrupt th~e expresion o-f a caene that noe a proteinroirdi a bik..nthetic -swas to a Ifirst aspect of iteeW Inveft ion. a Sa inoaeffc. rnicorgairsm has an Ettenvadni Iuato which drut heeer-ao of ;an a pparaus qene Locte'd witni the tipi2 ptoeiiy sa rdan inc mrerdent auxotrohic mato.The preorrd atensatngmutation is, w!t hin toe anaau ne ssaOV, anhe preferred axorohm riittonigaw'itfc s arcC:.or, :t~r The. mncoraim o'latyuter con-fpries n.o oe itrlg antiigens or therapeuticprcteins, hor e.~ml niatefomptoei E o hgf he-patiis A, 8 or Herpes Simplex, Virus and Human papiailoma vius Te reor. the miaocns a c sadlv. oie mus gis netosohrta 313The$&nnefa iicrc .nisnsmay b,,e us-ed i h mnate ofa r. Wicmn for imstaverlluu o, r wral zei;iveyrv Weti tresatteml oif a sitic Qrfatin e fg -r the, t aatrnentofthi.

The atenuted amoni~a iicrorgaosmsof the preSEnt3veM Y vaccneswhih srpnsnct stmulte aticsalas ellas ssteic mmuit ir Il VwVO OW&MOe the MirCowrarl~tf$ CO not Cak4e spiceen abscesses in an anirnal mooel. wherewas rhutanss with sinp' inutattns do This is a particular adwantace of the 4ouble muAnts as defined hri n~ofthe Inventiin SThe rnmcroorganisms and vacxrfle cornpcsi ins of the p resenw inventi on, may be prepared bylknown rechnique&, The choice of partiuar Salmpn e~erccoona and the salection1 of the aP propriate mutationj cn bei, MAdeS bV the sRle ers&on wibou, undue experimentatin, A preftmed macroranism is Sw )mflJ Inat~ a. oA firsl rtnuletion May I-st inlrobuced intoe an apparatus gene kiooaied within the reg*ofl of the $ah mnrie patbcypeniity isiand2.. thI~is regioni being disaed in The $aimnorneile psthcogenicity htlnd two (SpZ) is one of two ctassical psthogennity Osancs located on the Sknelchmsre.Spi2cmpie several 33 genestha encode a 'type 11 serto sse nvoved in -transrorthnc pi.. encded viutence-aessonated proteins (&C,-afld effector Pru-.is Outslice ottOle safrnoneie bacteria and votentialy dwrectly iltc Itarget host cells such as marpae.Part of S:pi2 (the apparatus genes) enodes thne seaueton apparatus Of the tyn~e Wi System Spi2 is aely essential for the paogrei nO virulnce of Mannt~ nte o mouse, an observation flw documented by severaldiffeen groups around the weld.

S. tvphxrnuriwn, Spa2 mutants arehihl attenuated in mice chlna d dve the oral.

intravenous anktaeidla otso atnminisraton.

me apparaus genes loc-ateid Within Spi2 are now well -tr r _k;d e: fo exrrmie Hensel &aAwl MWle-Cular Mirobiicy '1997); 24i1): 1;i55-167. Genes- suitbe 2 5 "F 6usc in the present inlventin include ssaV, ssa& sat ssa sskt seP, ssaQ satR, saS3, sse 7, ssWa an d saH oeig nes Thmutatlon in the Sprepion does not necessanik have to be withuin agene a my 4lso Ae sufficientv to disrupt gene- function- .n a pre ferred ernooirent of thE inventio.Drir Ie apparatus pe ner is sa V, in a enras referrTed erbarfirnent, thie muAtion les within, an intergenic regtosr: btween as)and s.,a.

WO: OMMO V/C %652t1PcT/CBW)0P49 which l esseni vn a tiosvntnetic pathway, The bi-nhe Ah-way j-s one pmesent in $Samnonfle but not present m marirais. Th-erefore, m;-e mutanits oaincn depenid ,on rrmtabc liter, ound4 in the treated6 patient. to circumvent the. affect of the mutationm Suitable a nes for tMe auxctrOpric' tmsatmmR include any am gne. ag, amk, aO, am d r.

"n a prefweret modmn of the invention, rievacecrntStflopss a $amionefta microraflism tiaving atterxua~g mutations- in, ssaV and amrC.

T he mutatinsm may oe introduced into the m ccrganis. UsingO any knwnf m0tchntique. Prla lte Mutation 'Is a delion mnutation, where diuotior of Ine gone ts ca use"d by treV exmiion of-nucleic acids. Alteratively, mutatic.ns may b~e Mtomcu-cd by the inselIjon Qf nucleic acids or by point mutalions. Methocis for introducmno the mutations irao the specmific reojons Mill be apparent the r*kilied pro0n.- Inm addition to Ine two mutationts, the Saimonefte rncroopalisf maey aiso cornpfise hetwaologc-.is antigens.r 'The attennuated micrOomnanmsm can theretore act, as a delivery vehlicle to dntseigantigens a gainst Other bac terial or Vvt inteciions.

Antigens which are suitable for use in thsway wi be apparent to~ tre skilled per-son znd include: S0Psthooenic E.V ~t,aligerx5 iL, [7EC MHepatiti A, B and C antigens Lime disease antigens vibrto cholera anticens Helicobacler anitqenis Herpes Slipex virus antinenls Human paplioma virus antigens This system aiohas the putential tko oiiver therapeutic protins. e.g, cytokines, for the treatmntn of patients, eg. patients ineldwith hepatitis. Methods for Vie del.ivery of heeooosantipens o, t'herap-Outic proteins iusing tMe vaccinS- 0 mpssitions will be apparent to +thes~e person.

Vaccines rrsrie using, the moogns of the invention have 2pplicationl Mo the rmnenl of infections in human pavents and in the treatment ofvtenv fections' The dou~rv muttiln Provide a efetve means tw ittenuatm the to, provide a sate vaccinle Qancdat~e.

The viccine com p05sstN$ provide effect"e protecto '0 e inrntncocrnprornYois~d patieants, Vld irnpan1 ttr 'I e. w risk i deve~apin2 Spl een att ~bcaIe S' 5. spleenr a h5ceSSe V ra been 16 ern fhed rsin p vacci ne s base -d o'n a s'mutabtrn, and tneeorec the presep onarn a offer a substatai, benefit ID patientshe.uon my presepl nt <I composition logetter wt any Sw'male pharrnaceutlaliy acPtabla aoadtuvent, diluent or exciperttL Suitable fonruwtl :os wlU be apparent w0 thC sk~,iliedperson' T f 0oMultASUS May be Mceveksoed for any suitabie means ofatra1.

PrMeeed a~aitai isVia the oral Or IntraVteflous MoUMS anC the Vwaoones ate iive The numbe ,r of microorg anismz that are reoujIrwed to o e t eseni Mr thb~ forrnu"ifls" can- bte ter-nt anooptirnisec by lte :iled pason. owever in ~enerai, a patien*tme nyl be a dr n ntstered apprxmtyLV I0~ F~spreferatly approxirralty I&IO"CEUs in a s Inge dosg n The loll,'owng Examples illustrate the i:nv*entiofl eamp'W I1 Th~is Example decrbs h pre*-paatin of a mutani strain designated ZHg wnShli -a s activity was huntan oral ty'phoid vaccine. The stran iS beried from the vFirulent S, typtti strain

T

v y, orni:y -solateO Ifr a case of typhoid The oerveo stanha;s a dketmnec mjtzatior within pijrA and scA, T2for the constrution of ZHB S, typlIiT y2 wa- ks oriin a Ily isows-ed i ro a n 'Iindiu al wi th t y phoid fe v er i 1 ,N ha s ben, as e d f or thie d ervato b oraf a lf re rs etd]t soic vaccineV Th e sv-tir wa S aImed from the PilLS natinal rulture- tzllecbc~n at Colindale. it was coteiied as a typh~~t At~ethe NOTC number being Bi Cloning the &~yph arc'C gene f roan S, typhi Ty? S. ytvT? y was recovered irOn> stockV anlO grown ovenight in Lu?'a Beneri" (LB) broth, The ciswere harvested and whole c0 DNA wasK prepared.DA frar aents typhi 7y2 DNA were generatat. by pal ciavo wih A rsto enzvmL- SauSr. and the rfes-ulng fragments were Iboated to BamH1 ci-lee pllC7h to oenemw a osidlibrar-y of S- ynliTy2 DNA using E. coib HUn3 as,, rpndpi'nt. To is baWe the DNA encoding aroC- from t1he S "yphi DNA, the cosmid Elbraqv was rit W 0 0 (V 692 6, 1 W AlPCT/CEOO Afl 74 9 ranschtoe r1cvh' ABt2&4S which htoUrs a Mutation in mhe ie'tnC gene aniv i& ,jeerIan.,>marornatt: sompounds for growth. The transdcwn mrjXtusra Was plated onto m ,iia meci IaMn roca: xon luds a-rd ncualod at 37. A number of soatd olnis ar oseve f Uwt oengh i te o These bacteria had presumnably arisen as a consequence of ccmrciemnn Of the aeoc muitation in AIB2&49 tb a cosrmd clone- hatlourtng the inta&Ct eeoC nEi irom. 0Cs nid QNA frmm onle of these stmrin wa. s murified. A EkbHindll fraqment froM This cosmid wads (clomed to PUCI8 to gIve ptasrnid pTAC2 whc asal t oolret h eetion of aroC in AB2S-4.deokiab tha t it contains the, SJ Iyota eeC gene.

0o Generation of a defined deletion of the d~one I S. fyp#P Ty2 araC A defined 600b,,p deeinwscreated wht he cloned atvC gene using PCR.

7he ofiionucieotcle primers usqcd in the PCR wvere decu :lsing the pubiished IONA seq"Uence -t tee S3.trwsli-o eof-le iAcc 27I5. The Ct A 'to the eC goe was anolified ftam pTAC2 usingprimers SPQ 0 NC. ano SEQ 0) ND 1, 'SE-0 ID NC.

2 5 3 annmeal.s to vectcn- DNA, SEQ ID N 0 I anneals to te5' egion of woO., The DNA 2'to the SMCnD gene wa s amplifid usinig pmmers, SEQ IM 114,, and SEQ, ID NO. I SEQ Mt NO- 4 anneal.s to vector DNA, SV) ED NO, 2 anneals to the 3' reptarn of aroC.

Th e re-sufting PCPk productsl her):ba siles inzorporated into the 6 engds to. ciiitate cloning. The frap tentsM. we're cmknac inlo t.Ie vecor pUCIC6 The firrl pasmid construct designated pMIIAC22 conrairis a defined deletion of1 eeoC (posiin 544 to I143 on a_ 4 .5 kb Kincdll traornrit. -te Ht-dfH) ?r4aomnri msiserted ai t Htdst of pLJC &EI A single Xt.ail Site is ars t cithesite ot the aeoC oeettom Introduction of the aeeoC mutagtion Into the S. typhi 7y2 penome The suioie plasrni pCVD"4A2 (Doni.nbarg Raper. lnfectan and Irrirnunity, Iq9I: 59 43v10-431 7) was, uzet& a vector to introdujce The eoc" ceelion i fto the Qeoorne o f scrypT Thet A .8tzb /HdW1 Iragment corieanio th eoC abon was1 ~oitedfrooIAC23 ard the en-s madle blunt by using the Stratagern DNA pvlishanq kit- Pleasrid pC.WVD-4Z2 was 4nreakriasm: Iby cilpeshorin with Senat Treated with 33 was isltdand benoteo oYCVC.21.

pYCVC,2 I hrasm iwaec.. 100 tvp?"T7Y2 by usi-.1 a standard eiectroporctiofl protocol T-he p_,lasmid was,- able to imtworal a into thie Ty2 geno me', following recr~mbintetween the horriolou Fegiiom~ on the, piasroid and thenfotfl tcO give ac:,IBr resstn nf rnantms. T hese trar'sf omlants_ containsed, a copy uf both I 00MO865Z6 b ogialwiF typ.e amoC and the de6eted n gene. Grownng thiese strains in the ab~sence of amiiinakowett for a siaond recorobirtzlkit envem Wo Occur Which resjwed~~ CVn loso h ~D42 DNA sequences and one cm-y of the ertsC gne, either the AWiityp cop orth defleed cspy, S ypl y2bteria wtich had vnderqone that second eomnainevent were iderrmtied ras amr-nollin -mnsiive denvatVes- whtrh w.ere *ble to grow irn. thv presenae of 5% suc.se ijpC^VD4-42 caries the _TaCB gene wohch vine n, explressed result n a siucrose-t' sentiSve peotp) Strmina that artminec only the dJeWtec aeC gene were mnaiafly identified as Str'ains that were una~ie to grow on fnrrai media. plates in the e-bssence of a supplement of mate. Compounds. The sinG ;eno.ype was boyrmdL using RO-R anaiysms Pnmers 'having SEQ IO. 5 ar d SEQ IID N(O. C-v q prdc of §S prm e wid type ;rCand 400-bp for the deleted arc_ gene, Sequetnce analysis of the resultng PICR proou_'Cts cofritthie presence ofn requjire:d deeinin 5 ind'Mdiuai isoiltes des~Qatad TYS. C7~CTS D -h and DTh'10. These stnains were s;tored in tWirobans vis W t- 7 Ca 0 for lono, tm- storape, Strain :DTYR waas cho-sen -vf furthser manipulation.

Inttroduction of an -suV rnutation into the t vphi arOC mutant DTYh, A 7.5 kb Pail fragment containing the sseV region of s, b'ph; was amplified from a total DNA preparation by vsing PQCR and ciloned into the vector pCRQ2.

0O dnrvitropen), The R'CPI oligcnurcieo tde pr I ,ers employed, Naving SEC 0 NO. 7 and SEC 10 NO. 5, were demsigned to the S. typhiwtitrn? SP12 seepierice. The rsultng piasmid construci was oles,:gnateo pThrSV2I.

plasriid mnnsnct Mo esn dlto fe ssa V aenie wasp denved from pTYSVZI by usi ng reverse ofientation PCR. PR'imers annealing to the 5' (SEC I CD NO.

9) arid 3' (SE50 10 NO, Q.O regions Of the v'38 V open reading frname were, designed to tMe S. ?vphirnsdum, 5i 2 sequmncre. A n A vkll resticli on, s ite was i ncorp ,orated into the reg'.C, n of e ac p rimer 1,ma XbSI sa e was nco.rp orsted in to SPQ I D NO0. 10 'The Xbelt sit e s erv es a s a' a f or the sxe V mu qtatlio0n So t' can b e d ete !te s y byv resmtrctio n analyss. The resuiq POR product wa"miiect diacezton wi th Avnl and the 3o baczbone piasmid mweasitm. purified followng agarose gpel electrophoresis. Recircuiensation of the resulting frwoments,, at Mhe Avhl stl* 'esgae the reIq;uir.'ec detton constru'ct PYCSV I. pYOSVI cointains a 15,5kb Pst! tregnmt with. a defined; I 594bp deeinwitin the ssz V open reading iram i.

IWO MWMV WO O~M~61 TIMDI 1749 The stiaidte p~esmid PCVD442 was, w&sed as a vector tri introdune thezz ssV delelon 'into thef- pe norne cif the- S, typhi Ty2 araC mutant DThB T re 5-.5kt" PO ~4qren cntinp he szc I -2tion was iso le d frto i pYDEVIk zrd the ends mnade blunt b~o trea2tMent With Merow DNA po~ylmera- se- Plasrnid pCVt44Z wwas ,inear -eC, by digestion ifth SrnaL trezted wit ki. line& pho sphcttase and ligated to the kn ended tt~~a.The required conructjo was isolated and denoted rYZSGV2 14.

pYDS\ 21 A was introduced into S, typ h/ D71Yl by tis~nA9 wie ctIropraton.

Anp""i&re-&-stant transormants were seee and t~hn orown mn the abswx*~ of a mpkizi in a lx 'w for toss of the pCVD442 DNA seqenescr one copy of the ss".aV gene, e-ither the MWIp, ,p h c 'td cop 5tzistht a tsararc; ti seoond recornbi naton e.vent were-.tntfe as arnpicilinrsensillve. us, e~tr t=Oonicns Slrains5 that had;- retainedonly the Ceteted "La gene were rdentified>J usi PCRanayss. ruaer, ,,ain S 'V NO II and SQIDN 12gv ml foryis Mer id in V Cp cr~ z~n ktn Nh. V*2 oae apmuc .2$$dofor ns wid ype ssa V and£Spfrhedeec aVee.Sunc in ioiidalisnotcte ZH2, 2111. ZIt. 217 and 119, STri ZN cas nosen fr manufacture of a CesmP master cell ank., Exainpi 2 This Examnple describes he pre paration of aR .5 iytimwumT Mutant4 stra dC es ignatec WTO Fwhicnh has vaccine actvrt y a gainst h usrnan ga mtroarits,. Th~e strai n ~s derived fromr. the known hum-an viuent S, .si~n t stan TML TML for the con-st-ruction of WCO TML was hqndl isolated fromn a pal:ent suffen frn atretit and wa eid =Mn tetoaneS of Dr Johln Stevens at Sirmninghars UNuesiy -Y S iyopF, lriseo Wr' w come R ase acvn Lab ora'xne s an. as,,igc:n ed a cWtu re rnufber, B T$ NT cufture was obtwained fmn RimrringnarnUvasr Geneati-on of a define-d, deletion of the cloned ryphimrnuwf tSsaV gene A oasmid (plasmid t 2, ShaNL2t-S136 93, 2593-259T~ was psneraked by otonin g a 7.5kb Psti fraigment isctlW.ed fromn S3- 0pml~m2 ifPC the Pall sUt of pUC 6. 8 V ms oSfined cenTraly on this fragrnertt. A pasmld OOtP, cortaiina defined adeIetion of theu sac V ORF was deivdonm ptasr-nid 7-2 by uit reverse Oientation PC.R Pr4irriers eeangto ie F (SECQ K) NC. 10ond 3' (SEIC NO. 14}) regions of the ss-!aV open -readng frane wero wasinad to theQ~ &t yph~nruhm Spi sequence, An AvrwI restrictinn site was ncrrrae inuc th-e 57 WO M6&241 T1DQI 4 region. of each pone nd an, XbaI sitew~Loroae into SEQ ID NO, ,io4 The Xt* site sevsas a, tag for the ssVmt tso it can be detecteld easiiy by restrction analysi:s. ThIe resuWng POR Prodoc-I was su ieed to &:gestion withA.

and the backoon'e piasrd ntrio ules purifie d llow-inq soaroze qet ei&eaophoresi$ s RecreuisatOflof the resulting fragments& at the Avtll istickyrend& gave Vriereprired deletion coflstfl-t &k-signated pMDS 1-VI. pMADSVI contains a 5.5kb Ps", frnavnenf With a defined 18S94bp deketh r wtthin the oswVcxs en reading frame., artAwiland a X-& re.meton, site are at the site of the dcelion, The suicidte piansid pCVD4,42 was, 'used as a vector to intrloduce the sssV 1 C dewletion into, ttsa gennorniz of S. ryphinut urn TML. Th~e I5bPl raprnent cnan the vsa V deletion was isoiated f ront pMCS 'IS and the antrs made N unt bty treatnelnt wit Ki.enow DANA polymerase. Plasrri-O sCVO44 wa'F~s linearazedC by gestior with Smral, treated With alksiline phoszphatat&e and ligaled to the blunt-eridedtrgret.

a. 5 DS 0 Was introduced into -15. oypM,-sufmr TAL. using cor~upot'sn. TFo t end Mie -onstruct wasP trnsmed into the E, cviszrain S17-1 A pig, The conjugationl waZs performe'd acuncrQ to standarrd pnocedurez. HiasrmiO pMDSV22 was a-ble to \ntrte n theTIL geniomre ft-AlioMg rcmiairbtentehmla regonrs onk theW alsi nd the genomne to give ampiciin r-esist ant rnortat 21 C A s 4U4d esi nale rncswAv7 wf oe or fugoe manigulations hi IrWnSCOnjug~att conaainS a copy of otn the cQhinzai WiWdtype ssaV and Ine de~eted saV gene.,, It wexs i~rown in the absence of aripicifiri to* a-IlOw for a secortcl Mecombinatian eventl to Dccur wtiCt -woud result in, the foIss, of the pCVE442 ONA s equel ces arid one copy of the sse gene., either toe vi -idype copy or Ve del.eted 2 5 copy, istdaies which had unaergone this sewnd recorobinatr eventl were identified 2,s RamPiC'lin-Sermitve csenvatives wthich were ale to grow ;n the presence of sucose(pGVD42 carrICs the sac gee winnviren exorezsed resu Ls in a socrosesestie phenotyp Strains that had retainerd only the deleted sa? or! we identified b. -usinq POCR analysis, Primers,- having SE LC. 1 5N :D O- leart nve a produdt otQA$Sbp for the wild type ssaV and $Sibp forte eltdSsaVoene Secunceanaystof te rsulingPORproducts -onirmedth rsceote reure eetiojn 4 pndividual *,solaes. ZZ H2~ 23 ZHZS Id n Zln26. I estri Ware stored. :n L.3 Plus 1%glycarot, a >50%t lotI onoutar 7n st-rae Strai:n ;ZH26 was chosewn -for furter Man.,aipLauon WO QkNi"20 WO Oft~t6I PTIG~~t~t749 Cloning the &typhmun rn~ arpC gene from S, tvphirnwtsrnm TMIL Geno mic D NA was t olztM-0 fromI S. yphmmunurnTh.i1 and mleave*d with H.qal~i fidi raqrnts- mf th§e size range Sut6kb were pur'5ied and at* W Hihd -iteaved r~luesalptk The iioat~orn mixture was used tc. traorm an, E. colt aroO mutant, A8/~2849, and clones ctafgthe S, tYphikivmamsC gene were selecte by virtue of their btyto complemente mh sa", Analysis of one dohne, pLACI. de-ostmreed that it, contained a 5..2kb 'Ih~ fragm ent.

A alptneci e00b-p zeei asceatd withr the rioned aoc gene by using tApR Th lgncetd rmr eedsrduigtepbished DNA sepuence i o c" S. typhi as-c gene ('Act, M277 1 The"r DItA 6i m, the ro gene was arn pliied frorm p DAC I u S ing prnemrs ang SE Q 1C NC>O I S ainn S ID N0 7 SFQ IDL NO., 19S anne am to ve ctor DNA, SEQ I D NO, IT- a nneals no th region al" raC, The* DNA 3' tc. tIn awCu gtene was arnpliffiei using porrers Z-0nSE ID NO, 20s and SEQ I NO' 0 10. SEQ 0D 40, 20 anneals to vector DNA, SEQ 'D NO. 18 anneals to, them3 emon W am-C Th reUtngP~CP products had Abela sitesnorrteinote' ends t4o faci:Itate ctonw{. The framrents vetsccnd noh erpC The final clasr4E contrut pMACScn ain ric ned elelfon of ,u715Oositiofl $44 to 1143)I on a 4.8 Kb, Kisdl fragment T1he 'Hin4II fraoment is insesteod at Ime.

findV site Oft PUJCI eK A Sint)le XbsI site is present 8 at h site If te -eltaon, i0ntroduction of the oroC mutation into the S. zyphhnv-lum see V mu tZ26 The suicide piesrmd pCVD.442 wwas uswd as' the Vect to ,ntMmduce them amoC ddeletvm into the gen ome of S, mohirnrimdr TML The 4.k AKiroI fn tragsnn containing the aroc, delenorn was 4 5cited firm pMP\AC& and the ends! made bkmn b ujsing the Stratagene Z-IN.A polishing Ki '%Part Nio. 20-040). Piasmiid pCVDAA2 was Li Lrie y d'Iipestion w~th Sminai, treated with aktepspfae on. iigated to ~the cdultndC ta~ens.The recui-rec constru=tw~e~stm e and denote pMCV'Cle pMV~ Swas ntroduced into, typhimandim 2H26 by sing electroport ,on.

Arnidliirras~rritvrastnrnersWaPre s elected and alioweC to' grow in the absenice of ainpicihn to allow lcr loss of mew pCVDi42 DNA sep"-Kenes and one copy o)f the aw0 ene either thev wildmtvce copy or heeldCopy, Sirans that b-'ad unOC gone tNus seconv rec-ombinatlon event-were dnt idancin~estv ewtva that we Nc' o tro grow in th e prsne Of 6 4t sucr:o Stwains, that hao relkineo only the delete-d araC gene Were initially identifieid as strains thM.at Were unable to c'row. on mnwial nre Ja platras Vnteabec of a su'polement- of aromaticcopud Th Vleo D(VOU] WO eOf6&~6'I PCT/C&W~N 749 aoSEO ID NO, 22 give a product Dt SS,4bu ior the wild type eroC and 400bp for thtN ad'elek! anC cie Sequenice analysis of the productQs c-onfirmed the oresenae of t'he reqired deleton in 4 ;ndivduei sotates desionated WVTc5. W4T79, WTIO and WilT2 Strain Vv'T0S was C~hosermfor Manufacture of a CSMIP master cel bank., Examzple animal modtel, S..tp SL'l$44, a strain that inlects mice, wvas used, with s~qeand duue mutatioms, -resent' Ar: ssaV.,-apfl norjvpoian' routlirn from S. ?vlptsi4)W)Wfl 1202:35 wasm P.22 transduce d to SLISA to give the -stgit SP2 mutant, The aroC deoaC suicide vetrpkACC ti selcoorae it tne S. tyhirnwnun straink Wl501 C and merodiptoid-, wvere obtained. The arc)C C)eetin mainrodipidi~ was Me p'2lzmue rom the' 5 WE~ meodpk to $LI The St 13,44 merodioid was thlen reovdus.tnq Sucse seeionl to gie the singla aroC mutant, -the double mutant was. generated ty P22 trensductton of tmearCelto matodiploid from LS5OI into the, SL134,4 xaai lrdsqecswr resolved fromt the rneadjpiold' 'ievitg strain, S. the amoC deletion mutation in, the SbI 1344 ssea V:wept baclkground., Pre-ckinicaipameWnai tde on defined olsVSln ei uat 'Salmor'&Ua mujtants {Strairs SL.1344) nanoving defined muwtation in either aroc smav oV a" ooibinatkcn oI botn mrutations have been evaluated exlensvvein BALB/0 mire to assess attenualion, persistence 0f the aroanJS'm and abiiity to irnmunise against m:ha :enge witn vhre wid type Example 4 Animals, inwnised by the intravenous route Protection studies Z~czupsof ter, BALBS/C mite- were: umu ist. wth 10G' arc I0 D" nism of SL1344 crGS 44 s-saV,. and 3L-13144 arC saV grown overmotort in L trot I 0 wil type or nanmsms te'en kitavenous YL Ten o-rcanisms oflthis wil type strain,, given, intravenously are suffictent to kil truce.

PCVCGR0AM7 49R All the, m pegver. the sinpile PrroG or !ssEV mutantswere so'mddiy protecteri1 a-tte chaiienge with veler Oose and remained WeliTroghu the exoenMrnt, eKNNn no sn of Isea'sa. Fr the double mutant 90 f tMe animaswr oil rice that recev'ed the immuniisaticn with I0* organisms., One off the animal s diad8 d ays after Vshe che-lenpeL For thle rm lW thtWere- rmurised with the lower dose, Ony 1 of The rmce survved the onflengte.

This expehmemiceosae tlhat irnirsaxion Wih Sahnoneiia Ssav rrtutart, tither, atne. orin combination withi an anC mutation wil imrnrse mm apainst challenge with the wvil type Sal.ftnn&Ia- strain.

0. Persistence of Strains Grou~ps 0! mice were given IC )t rogaru.sms of the tnree Sahmflneia mu-tamts escribed- above. Fo-ur Mice wa crficeti al differet tine points up to day 14 ar enueraionof orqanierns in livturs and spleenz were pern'vmed. Counts of aW1 three mutants were. comparablie up unti dken uwnaritlhe c-ountweeapxitlySXtas organsrns in each organ, At day 14 a diffference. was ciionteedbtwe he "'nume mutants an'd the double mutants, there being a kog less, in Ine numbers of doutie mu gam or nss both liver arnd Spleens, The- other impomrien dferencle beotwsaen Me stgie mutant arnd';e amc' ssaV double- mtant is that lhare were no kveNr abscesses presen at any t ume Ounng The expermenjto th ,J i.,mtniHovvr h- ak inetdvll' leSns flid h~ave liver abscesses present at da:y 110 an d 14. This EiS an important finsnganostrrpl sPPOnsM thee of this combina 'or; of nultuOns for evaluatiOnj the nre'oaration of vacclrnes.

Pmmunocqencity 2, Mice. rnmnuniseo a above Were bled anal the antibody filres we ,re determined at~~airs. woecf$4noneBa using an -LUSA, All three strains were demonstrated to be highly in'miunogenic:, ei Ng h it-r of iulngIgOS against Salmonelia, Aqinials inrounitmil by the ana l ue 3 Pesitec of strains Groups< of niuce immunkised oraffl'y wIth 5 x 1 V, oranisinsv of each oW thc. three $atron atk mt ants were sauiia," Periodiic i-iteras- and the n-urmbers of Scamv trul'an c-ounts in, i vers andspleens were 1&I and I0rsetvl u p until about WO $IM6U6 PCHMMI day 21. Thiereafter the nvmbers reduced, ror tne rnice that received the aruc: dooeMutnts, oranms were v kjl ndete jctable. n theiivers anti np~eens after orai irnirsfiofl.

OWa immujniszt~on e3nd intravenoustl obaienge of A'-J mice vaccinated with: $WrS&IoneWl 4'phin.fvrnm TML arcCfssa V (WTO$).

The purpoe of: thi;s exptirtieft Was in asoenst n the prot-ectivk Icayo rCsa vhruu TML mutants in an, oral aty nrn vaccination anid 'fntraverious ciailersoe mnodet_ Th).is mnocci more 'Ish eeoestehmnr

S

to SWftnonefle in thai these annmals are iess susceptible than an it' bO~akpround.

It S 0~ S 4phi, Wun TML aroCs=V in a c'um o 02 ml PBS was inoultedowally ty qav45e tube in=,o 10 658%week old AUJ mitend left 8 weeks, w rite were t,,van ,S oNly wn tis time and serveas conr animals, After 6 weeks had kiaps'ed be two imminmsed groutps were: chlesttntraven:>Uszy with: 10' wild type S, ty pho-nwfimnTML Mice Were ob.ser'ved for 30C days Port &ha~enae.

M All MI animls e sofidly proteted aqainsil wild type C.haienue (lOO% surviva' 10/40 anml ai koe giveni P55 on and then. chailelno.et with ,Wtd typ twyp ninvrkj, TML die._d o n a&y 6 poasti ehalnege, Mn an iIY be ckground the doutble typhriuhuz T'ML arC/ssaV see=m to proteol Mice given anl oral dose oftS x I1C-5 This may b,,e important for the human C, situation as it mice are a betner Model -o rolman SaiineIU S, in terms of~ St-udies were a mjed~ ou W. eiv use the ~.ncoiedouble mutarit in the livers and splieens of the mitse. ft was, found thatW toe double mu;tant s pars t a low ievels to around day 21. ,y day 2$,i t mutant strain, has wenceared.

Exampie 6 Human clinical tr6al stuy kohwing appropriate screen. g each of Zvlnesreevdasincle ota ,dor of eifer W,2 1 0, or 101& CrUs of S, z'phi ZRS or S.Iplmru TS he 3 o mroranszm prepared as above were resuspende oteCpcfaedsA conenraion i afinal volumeci lof"tni of 2% (whi s odiurn Ltcamofiate solutlion neutrai2e gasino acid- This iiid suspensiorl was acm ni-wr orzlly t. thle volunieers. 'The volunteers were then isolat f or 72 hor.ari ben f rllowec up p0.51 irninunr~is tfl for Safely arnd snnroeni W( OAM?63 PCMG.00010 IM19 Volunteers were asserct for rc oncf 0jother adverse eventS asoizalad 'with vaccination by obs "C"eration, phys:d xamiato and by~ tjrhe C-0 o M Of 4;b iry Cards, W, acdciori. bkxood stool andI ulse Caltars Were colhecwed to assay for vpzc naerni, shaddinga an pa4 PMstfitce ot he vacctn str-aifls, Addijorr4 safe-lt dte as otaied b mesurng &eet of ac te protein-. (CRP?) ana hv er ksnoton enymws kALT) in: blood, total WINvte btood cel (CV-l. C waon-ts and evrlrocyie samv eztor rates {ER sgsadr rcd~.ThnEs prameters were measured on blood talken daisy unil day If and then at weekly rnevuntil day 2S.

Analyshs of nucosal andytei immune ftsponss ood nd slivasarnles ere dllere~ nortc immmntzaw~n ancl then on days, by EUS.A Pen ,perl 6tood mono;nuclear cell were :oIlecteo and assayed. for the presence of antibodwsecreting meIs(A- s using me ELISRST teannique t$Ct, _.4Vph t l ZH9 aML-S. wyrsnni AW15 were well toerate.d ink all of he, ISvolunteers No senous-adverse events were noledO in any Cof tie VOlunzme at each of the dose levels and blood an untie CulTUres remained neatvi ak v.am-ines ct c~l tmepoints exatrned, Thas, irnmunisatlon'AtIP- S. ph: ZI- and' S t~pinw am 00 rot asu to actaerie&None of the &OUnteers- given efther of the strains developed dit~roeo. persistent hig: ,grac fever, fute dsaatfiri the saf ety of the vaccine' strains.

Pesaeteceonnrvcnarabynca7wsnoobevdieteroth3 dose grouas of S" tvrniZtf&or in the lowds 0)o S rvpit.nnunWO ?Aucosa a ;Ind systemic immune responses elicfted by S, ryplhi ZH$ Oral ipnimrren with a singole lo w e xl 03CFs of S. rv Z H9 resulted in the phrring of S, -vfAspcfc 4scetmi~ 1n2 o, !o-uteers detected day 'Wter imnmunization, Subsecuenti testng on annys 14 and 21showedtll-ail rr ASCS were stil trtalebut at MUCh lower leve s cnd Pad daperdby 1dv 22. in alotall mspo denvacdnees, nun .bers of AS C-t wer highest on day?., Surprisinpiy inetino a higher dosie 10 t' CFIJS) e? S, fyc rI rslt-d in a low- luA ASC res-pons-e in oly one of -tree vac-'inees. ingestori of toe nighe'-st dose ifl~OF i primed bqA ASZ~s in 2 of Sc lnonlla~pacji-Serum antibody response Oral irnunazcton wit z sorTi'e iuw nsfe Q" xiS>" CYtLIM of Oypihi.ZH file~d to elci S r~d L~sedicSerum lgQ espite gekrtn gA AS s 0n, vecelneesi ne examied onclays 7, 1"4, 21 aod 26, Simil arly on ,ly I poce vry low, levels of fllaueilaboth LPS and gl pfIgGl~ in all 3 vrlers. increased els. of Ir Prs ,p--carnd tlagella'speific were detected as SaidY as 7 ciavs after vaeziration F ising on day 1:4 ancd remaining hiMn e hes oeo1 C sa' tiutdLP' flap or sn 7a and 14ri-tvey mmd ii ta-specifc IgO in 2 of 3 vaccareesd cad an days7ad4repciey contlusions This Study damonstrated util' w of ~v~mtatiofl. as a a-miponenl of any new Oral typhoid vsctea sutn- An, &yhs stramn haturin am mutAtions alonwe Wvuld, 1 have ca~secl va. aerims W the doses gher, The ssaV mutation tieref ore pmo or..a 2 0 eclitonal ievei of safey to The am,~ mnatl$on aMon, by alldn h v04iaf~5 If !Ns early forrnilator.

As we as prvil0t be welioieaed 0 ZI-9 was also demonstrated wO be ntamboate(i~ ZFs) tctlihes CF~ um~mrprved to be, higpny Irnnunogenl.

as ith S13 veales given '1C0 C and oiven IT CFLVs &'itdtg la ig of b4& typN0"LPS and fiagelia specifc-serim lgG- 7hese repsponses are vwery enO0Ur$9Irg sinmce is enr~lydifiul t eic seut nioy b>y oral vaccination.

As m wel sonerabnfl sr PhspedfiZsenral rnu0Oy responses, Z.HS also &ne4w, ,9A A$Cs., indictive of immune stwmnuleto N at te intestnlrncs ttlo w.unteer eficited n SpL$ tfi cAartn el{S) apnC ictdt o -Mr*,rto be dose-dependentl W~Swa alsz well tolerated ano no vacanias wore aetacted, ksteres1%v*y no, Crho5 or symptom s oi gasromentets 'were- detectdi no ouves h rcevious date Obtained uswn; the rutant TIML mtran YWi s~nir s aro 1rSI2 mwmhAetf Ir, $4~~tninum~iventc mice suu tested hateaduti aofsrmt might case sm loalnt"bna! effects e~g. ciarme& -Camps intiumns FT The a bsienrce, ofm i> s:re eve,, n t further Supports tri utility oW thez =omnbineafl ci af m andi £21 mutmans.

a0To dcrinlaethe tliyof tie ssa V -a c duble mu tant strns to express andi deliver fsI bn ant.sqenIsa WT0 Ws trnstfirx sri th a plasma (pBERDCD2) expressing The- gene for The C. cots' it-leojie cintercmOnfl B subunik (Li-) B L RIC mnice pn;= I Zt .pro)u p) werTe3 i mmimu r ize d carislty,,,-ic In days 0 and 25 with. "I 0 C tF Us ,2OCTnl in PFTS), WICS axpreszlnq pIRD026. orwite VVT05 veiz.cr str (commelD. rom ccrnpaisn(ads positve WInro) a gtvup of Mice Were inimuwnised Drally On onays L0 art 28 wiM 10 p.9 jrfed LT (Sigma). Negatve cnr mice were wmns CjyOn clays 0 and 2$ ,WthZ DD0 p PE&, Mice were.Nied "rM the ta vein of'. days. 21 28, 3$ ar34I by Cad= uctr oft oavy 42 and sara arid inteztirWi iavage (day 42 Only) coillec&O1 an-d starad al, -0T AM but onte of tht nune r--nn d with WT3$LT-Z$ .eicted L71 specifti G (titss of day 2S afte a tir le Oa; iose Nonle of the CntV micintnurec expesn~paIFD02$t eftiud amrost axisvLyThspecific l9G~al, indizatig a tIA towazrcs TM741type mrune response. 4n ont-ast n mce nm&- win punred LT {Si na} eId aimost exctxsively LTspeftc lg~G1, inldicatini A tM2-typw t rsponste.

Therefore. expressing the LS w- ~irt vts enCi Ssa v strnfclttsrooii immune a. notaton Thie THl4Aa sed raspan--s usomane ~d bythe Samnlaaoist str wI be irnpvrwnt, wbe" antguens fromrp~cekrairafo ~~d43 epne are Protective, are expressed.

Claims (10)

1. A Salmonella microorganism having an attenuating mutation which disrupts the expression of the apparatus gene ssaJ located within the Spi2 pathogenicity island, and an auxotrophic mutation which disrupts the expression of an aroC gene.

2. A microorganism according to claim 1, wherein the microorganism further _comprises a heterologous antigen or a therapeutic protein. A microorganism according to claim 2, wherein the antigen is a hepatitis A, B or CC antigen.

4. A microorganism according to any preceding claim, wherein the microorganism is Salmonella typhi Ty2. A microorganism according to any preceding claim, for use in therapy.

6. A vaccine composition comprising a microorganism according to any of claims 1 to 4, and an adjuvant and a physiologically acceptable diluent.

7. A composition according to claim 6, comprising from 107-1010 CFUs in a single dosage unit.

8. A composition according to claim 7, comprising 108-109 CFUs.

9. Use of a microorganism as defined in any of claims 1 to 4, in the manufacture of a medicament for intravenous administration for the treatment of systemic bacterial infection.

10. Use of a microorganism as defined in any of claim 1 to 4, in the manufacture of a medicament for oral delivery for the treatment of systemic bacterial infection.

11. Use according to claim 9 or claim 10, for the treatment of typhoid.

12. A method for the treatment of systemic bacterial infection in a subject comprising administration of a microorganism of any one of claims 1 to 4 or a composition of claims 6 to 8. rcrcI'wCl1 ?49 WO) t z*il MUZc- N)ICEti$3N O' P220 pt~2ae 0 6o "1 Zrn~d of O <211 NC2 2100> MI WO) f6961 21 2 a e-c Z- pt- .nC Art IC~ <21 "1>2 <Z12> t <211 2. (4 U"0> S DNA wo W69161 P(--pcHa&Ta -j,4 ,k .2Z 7 <22Z2> DtNA -0 >A-~C3 <ZZ3 0 f 0 Z2a 6? 2 2 C' k r.fr>I evn~ gk t c c <400>DNA 2 x 11k i 4A ie S 4. Cnce 0 g eC '222>P t~5e~fscrptQ of .A.~ti ac~im2 Ssr~a 71 cCei cQ~-m2Aeoti <400>gr cz 00p9S~ 6 VVaO W2 926 -2 <212I> 230 s I Q TJ- ir t C~Z,~tCl ep~aCZ&$S.P9 fl3ij: ~q.~\4.:~.~TUaOFI <t C3ue z4-tt-Dt~6tcgt 366 cl~~ 3 a§yy6~ Ct p wo 00MI.61. CZ 0WK6 1KYCQWX'4 e~~2 £T act 3 Nr' Be ccU aene <2 c r~ .ce ~ec lyn 4of