CA2045842A1 - Dna capable of site-specific integration into mycobacteria - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A DNA for integrating DNA into a mycobacterium chromosome which includes a first DNA sequence which is a phage DNA portion encoding bacteriophage integration into a mycobacterium chromosome, and a second DNA sequence encoding a protein or polypeptide heterologous to the mycobacterium in which the DNA is to be integrated. Such DNA may be integrated into mycobacteria, which may then be administered as a vaccine and/or therapeutic agent.

Description

~T~P424 ~J~

D~ C~L~ OY SI~- SP~CI~IC IlmG~TIOII

This invention relate~ to DN~ capable of integrating into mycobacterial chromosome~ More particularly, this invention relates to DN~ which i9 capable of ~ite-~pecifi~ integration into a mycobacterium chromosome wh$1e containing a DNA se~uence encoting a protein heterologou~ to th- mycobacterium in which the DNA is integrated Certain mycobact~ria r~preJ-nt ma~or pathogen~ of man and animal~ For ~ampl-, tub-rculo~i~ is g-n~rally causet in humans by MYcobacteriu~ tub-rculo~is, and in cattle by MYcobacterium bovi~, which may alJo ba tran~mitt~d to humans ant other animals MYcobacteria l-Prse i~ th- causativo a~ent of leprosy M
tuberculo~i~ and mycobact-rla of th~ a~ium-intracelllare-scrofulaceu~ group (MAIS group) repr~ont ma~or oppor~unistic pathogen~ of pati-nt~ with acquired im~un- d-ficiency syntrome (AIDS) M ~-udotub-rculo~is i~ a ~a~or patho~n of cattle On th~ other hand, Bacill~ Calm~tt--Gu~rin, or BCG, an avirulent ~train of M bovi~ widaly u~-d in hum~n vsccines, and in particular i~ u~ed a~ a liva vaccine, which i~ protective again~t tuberculo~i~ BCG i~ tho onl~ childhoot ~accine which i9 currently giv~n at birth, ha~ a v~r~ low incitonc- of adverse ~ffect~, and can ba u~-d r~p-at~dl~ ln sn individual (e8 in ~ultiple for~s) In ddition, BCG and oth~r ~ycobacteria (eg , M ~o~ ~tl~ ~plo~-d ln ~accln-J, ha~a ad~uvant propertie~
amon~ th b~-t curr-ntl~ known and, th~refor~, stim~late a r-ci~i-nt' i- un ~Jta~ to r~pond to antig-n~ with gr-at ffactlv-na~-It haJ be-n ou~-oted b~ ~acobo, e~ al, ~YE~ Vol 327, No 6122, p~ 532-535 (Jun~ ~1, 193~) th~t BCG could be uJ~d as a hoot for the conotructlon of reco~blnant ~cclneo In other word~, it wa~ ~u~ tcd to take an e~l~tln~ ~accine (in thi~ case \

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again~t tub-rculo~1J) and e~pand it~ protective repetoir~ through the $ntrotuctlon of one or more geneJ from other pathogen~
Because BCG vaccine~ are admini~tered a~ liva bacteria, it i~
essential that any foreign anti8ens~ polypeptides, or proteins e~pre~sed by the bacteria are not lost from the bacteria subsequent to vaccination Tran~formation, the proce~s whereby naked DNA i9 introduced into bacterial cell~, ha9 been carried out succe~fully in mycobacteria Jacob~, et al (1987), hereinabove cited, have describet tranJformation of mycobacteria through chemical method~, and Snapper, et al PN~S, Vol 85, pg~ 6987-6991 (September 1988) hav- deJcribed tran~formation of mycobacteria by electroporation El-ctroporatlon can glve fro~ 105 to 106 trsnJformantJ p-r ~g of pla~mid DN~ ant auch plaJQld DNA' 9 may carry g-neJ for re~l~tanc- to antibiotic ~ar~er~ such a~
kanamycin tSnapp-r, et al 1988) to allow for selection of tran~formed c~ ro~ non-tran~formed cells JacobJ, et al (1987) and Snapp-r, et al (1988) have also de~cribed th~ UJ- of cloning vehlcleJ, Juch a~ plaJmid~ and bacteriophag-J, for carr~ing g-neJ of intereJt into mycobacteria Combination of th- abov~-mentioned techniqueJ, along with ~tandard toolJ of ~ol-cular clonlng (~ g , U~Q of reJtriction enzym-~, ~tc ) allow~ th- clsnin~ of ~ene~ of int-reJt into vector~ and introductlon of such 8-neJ lnto mycobacteria To ~pr-8~ th~- g-n ~, lt i~ i~portant to hav- availabl- slgnals for g-no ~spr-~Jion, in particular, tranJcription promoter elem-nt~ Such pronot-r elem-nt~ ha~c be-n i~olated from mycobact-rl-l h -t ~boc~ e-n-~, and used to e~presJ foreign antig-nr ln ~cob-ct-rl~
Pl-~nid~ curr-ntl~ vailable for u~ ln ~reobaeteria, how-v-r, ar- not t-bl~ ~-int-lnod, and ar~ readll~ loJt during non-sol~ctl~- ~rowth I~ r~eo~bln~nt N~cobact~rla e~pre~lng gen ~ of inter-~t are to b- e~ploy~d a~ vaccine~ againJt a particular ntig~n or a~ a m~an~ of pro~iding a th2rapeutlc agent to a ho~t, which i~ expres~et by the mycobacteria, it i~ crucial that ~3uch genes not b~ lo~t from th~ recombinant mycobacteria sub~eqluent to their administration It i~ an ob~ect of the pre~ent invention to introduce into mycobacteria and ult$mately e~pre~ a variety of heterologou~
genes, including, but not limited to, gene~ for protective antigen(s) for a variety of pathogens, and/or for other therapeutic agent~, th~reb~ enabling on- to protuc~ effecti~e vaccineJ aKain~t such patho~en~ and/or to providc effective therapeutic agent~, whereby such g-n~9 will not b- 109t ~ubJequ~nt to vaccination or admini~tration o~ thc therapeutic aBent .
In accordance with an a3pect of th~ present invention, thcre is provided a DN~ which compri~- a firse DNA s-qu~nc~ which i~ a phag2 DNA portlon ncoding b~ct-riophag- int-gration into a mycobacterium chro~o~o~-, and a 3~cond DN~ ~-quonc- encodlng at lea~t on- prot-in or polyp-ptit- wbich i~ hat~rologous to the mycobacterium in which th- DN~ i9 to be inte~rat-d The term "phag- DN~ portlon", a~ u~ed herein, mean~ that the DN~ sequ-nce i5 d~ri~d fro~ a phag- and lack~ tho DN~ which i~
requir~t for pha~- repl~catlon ~ actoriopha~-~ fro~ which the ph~ge DN~ portion ~ay be tcri~ed includ-, but ar- not li~it-t to, mycobacteriopha~es, such a~ but not - ll it-d to the L5, Ll, Brbl and TM4 mycob~ctor~oph~ ; th~ la~bta ph~g- of ~ coli; tho to~in phage3 of C~rvn~b-et-~la; ph-~-J of Actino~c-te~ and Norc~tia, tho 0 C31 pha~- o~ ~t~ ~toR-c-~; and th- P22 ph-~- o~ 8~1~on-11a Pr-f-r-bl~, th- ph-~- DN~ portion eneod~ eob~ct-riopha~e int-gr~tlon lnto ~eob-ct-riu~ chro~o~o~
In a pr~-rr~d e-~odim-nt, th~ flr~t DN~ ~qu~nco include~
DNA ~ncotin~ int-~r~--, which i~ prot-ln th~t pro~id-~ for \

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.J, integratlon of th~ DN~ into th- mycobacterial chromo~ome Most preferabl~, tho fir~t DN~ sequence al~o include~ DN~ which encode~ an AttP ~ite The DNA sequence encoding the AttP site and the integra~e provide~ for an integration event which i~ referred to as ~ite-specific integration DN~ containing the ~ttP ~ite and the integra~e gene i~ capablo of integration into a corresponding AttB site of a mycobacterium chromosome It i~ to be understood that the exact DNA s~quence encoding the attP site may vary among different pha6es~ and that the e~act DN~ ~equence encoding the att~ site may vary among different mycobacteria The integration event re~ult~ in the formation of two new ~unction ~lte~ called ~ttL ant ~ttR, each of whlch contain part of each of ~ttP and ~tt~ The in~ert-d ant integrated non-pha4e DN~ which include~ th- fir~t and s~cond DN~ ~equence~ flan~ed by the AttL and ~ttR ~ite~ Th~ in~-rtlon and integration of the pha~e DN~ portlon re~ult~ ln the formation of a transformed mycobacterlu~
ApplicantJ hav~ found that, wh-n employlng the phage DNA
portion oP tho pre~ent lnventlon for integration into mycobacterial chromo~ome, th- gene(~3 of intere~t which i9 integrated lnto th m~cobacterlal chromosomo 1~ not lo~t followlng non-J~lectiv- growth of the ~rcobacterla Thu~, the gen~(~) of lnt-r~t ~ay be expre~ad b~ th- mycobacteria following uch non---lectl~- ~rowth, thu~ ma~lng ~uch tranformed mycobact-rl~ ~c-ll-nt vehlcle~ to be omployed ln vaccines or pharmac-utlc~lJ wh reby ~uch mycobacterla wlll e~pre~ anti8en~
and/or th-r~pautlc a~-nt~ of lnt-r-~t ~ub~oquent to adminl-tratlon of tha r-co~blnant mycoba¢t-rla to a ho~t Mycobact-ri- into whlch tho pha~- DN~ portlon may be int-grat-d lnclud-, but aro not llmlt-t to, MYcob-ct-rlu~ bovl~-BCG, M ~-D~atl~, M ~Ylu~, M ohl-l, M fortultus, M lufu, ~
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Paratuberculo~i~, M habana, M scrofalaceum, M lePrae and M
intracellulare In a preferred embotlment, the DNA i~ integrated into MYcobacterium bovi~- ~CG
The second DN~ sequence which encode~ a protein heterologous to mycobacteria may be DN~ which i9 all or a portlon of a gene encoding protein(s) or polypeptide(~) of intere~t; DNA encoding a ~electable marker or marker~; or DN~ encoding both a selectable marker or marker~ and at lea~t one protein or polypeptide of intere~t ProteinJ or polypeptid-s of lnterest, which may be encoded by the second DN~ scquencc inclut~, but ara not limited to, antigen~, anti-tumor agents, enzyme~, lymphokine~, pharmacologic aRentJ, immunopotentiator~, and reporter molecule~ of intere~t in a disgno~tic conte~t Antig-ns for which th~ 3econd DN~ ~equence may encode includ~, but are not li~ited to, MYcobacterium l-Pra- anti8en~;
M~cobacterium tuberculo~is antigen~; Rlck~tt~ia antig-n~; malaria sporozoite~ and merozoit~s; diphtheria to~oits; tetanus to~oids;
Clo~tridium antigen~; L-ish~ania anti8enJ; Sal~onella antigens;
Borrelia antigen~; M~cobacteriu~ afrlcanu~ antigens;
MYcobacteriu~ intracollulare antigen3; MYcobacterium avium antigen~; TreDonema a~tigenJ; PertuJ~is antigen~; Schi~to~oma antigenJ; Filari~ antigen~; HerpcJ viruJ anti~en~; influenza and parainfluenza vlruJ antigenJ; meaJle~ ~lru~ antlgenJ; ~umpJ virus antlgens; hepatiti~ ~iruJ antigen~; Shi~ella antigen~; Neisseria anti8en-; rsbleJ ntl~enJ, polio viru~ anti~cn~; Rift Valley Fev-r v~ru~ anti~-n~; dengu- viru~ anti8enJ; m-a~le~ virus anti~-n~; H~n I~mNnodoficl-ncy Viru~ (HIV) antlgon~;
re~pir~tor~ ~Jnc~ti~l viru~ (RSY) ntl~en~; ~n-ko venom nti~ons;
and Vibrio chol~r~ ntig-nJ Enz~me~ whlch ma~ bo encoded include, but r- not ll-lted to, ~toroid ~nzymoJ
~ nti-tu~or a8ant- which may bo encoded bJ tho ~cond DN~
~equ-nc~ includ~, but ar~ not limitea to, int~rferon-3, \

interforon-~ or int~rferon- ~ and tumor necroJi~ factor or TNF Lympho~ine~ which may b~ encoded include but are not limited to interleukin~ 1 through 8 Reporter molecules which may be encoded include but are not limit-d to lucifera~e B-galacto~ita~e ~-glucuronidase and ca~echol dehydrogenase Other peptide- or protein~ which msy b~ encoded by the Qecond DNA sequence includ~ but are not limited to those which encode for stres~ proteins which can be administeret to evok~ an immuno re~pon~ or to indue- tol-ranc- in an autoimmuno di~ease (o g rhoum-toid arthriti~) Sel~ctabl~ markor~ which may b~ eneoded includ~ but aro not limited to tho kanamyein resi-tane- mark~r the noomycin re~istane- mar~ar th- ehloroamph-nieol re~i~tane- mar~-r and th- hygromyein ro~i~tanee markor The phag~ DN~ portion of th- pr-J-nt in~ention which include~ tho fir~t DN~ ~-qu-ne- eneotin~ mreobaet~rium phage integration into a myeobaeterium ehro~o~o~e and th- ~eeont DN~
sequene- eneotin~ t loa~t on- protein or polypeptide heterologou~ to m~eobaet-ria ma~ bo eon~trueted through genetic enginoerin~ teehniquo~ ~nown to tho~e ~killed in tho art In a pref-rret e~bodim-nt th- phago DN~ portion may bo a pla~mid ineluding in adt$tlon to th DN~ neod$ng integration and the DN~ eneoaing a h-t-rolo~ou~ protoin an origin of r-plieation for any of ~ wit- ~ari-t~ of or~anl~m~ whieh ineludeJ but i~ not li~ito~ to~ eoll, StraPto-neo~ sp-ei-J B~eillu~ ~peeie~
Sta~h~loeoeeu- p-ela- Shi~ poelo~ S~lmon-ll- 9p-cie~ and ~-riou- p-el-- of pn-umoeoeel Mo~t profor-bl~ th- pla~mid lnclud-- n orl~ln of r-plication for ~ ooll Th- ph~ N~ portlon l~o m-~ lnelud- ~uitablo promot-r Suit-blo pronot-r~ lneluta, but ra not llnlt-d to, q eobaetorlal pro~otor~ ~ueh ~- th- ~ca HSP60 and HSP70 pro~ot-r~; m~eob-etln promoter~ of M tub-reulo~l~ and ~CG tho ~up-ro~lt~ dl~muta~e \

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promot-r, th~ ~-antigen promot~r of M tuberculo~i~ ant BCG, the MBP-~O pro~oter, th~ 45 kda anti6en promoter of M tuberculo3is ant BCG; and th~ mycobacterial a9d promoter; the mycobacterial 14 kda and 12 kta antigen promoters; mycobacteriophage promoters such as the B~bl promoter, th~ Ll and L5 promoters, and the TM4 promoters; E coli promoters; or any other ~uitable promoter The ~election of a ~uitable promoter i~ deamed to be within the ~cope of those of ord~nary 9kill in the art from the teachin~s contained herein The promoter s-qu-nc- may, in on~ embodim-nt, b- part of an e~pres~ion ca~-tte whlch also include~ a portion of the gene normally und-r tho control of the promoter For e~ample, when a mycobacterial HSP60 or HSP70 promot-r i~ employed, th- e~pression ca~ett- may includ-, in addition to th- promoter, a portion of th- gen- for tho HSP60 or HSP70 protein Wh-n th- e~pr-ssion caJ~ett- and th- DN~ encoting th- h~t-rologou~ prot-in or polyp-ptid~ ar- e~pr-~-d, th- protein ~pr-~s-d by th- ca~sette and th- DN~ ncoding th- heterlogou~ protoin or polyp-ptide is a fu~ion prot-ln of a frag~-nt of a mycobacterial prot-in (eg , the HSP60 or HSP~0 prot-in), and th- h-t-rologou~ protein In a pref-rr-d bodiment, th- transcription initiation ~ite, thc riboJom-l bindin~ ~ite, and tho ~tart codon, which pro~ide~ for th- initi-tion of th- tran~lation of ~RN~, ar- each of m~cobact-ri-l orl~in Th- ~top coton, whlch Jtop~ tran~latlon of mRN~, thor-b~ tar~inatin~ ~ynthcsis of th- heterologous protcin-, and--th tr-n~cription termination ~it-, may be of mycobact-rl-l orl~ln, or of oth-r bact-rial origin, or such stop codon ~nd tr n~crlptlon t-rnin-tlon ~lte may be tho~a of th- DNA
ncodln~ tha hotarolo~ou~ prot-in or polyp-ptid-Th- ph-~- DN~ portlon ma~ b- emplo~-d, ~ herelnabov~
indicat-d, for int-~r-tlon into ~cob-ct-ri-l chromo~ome, th-r-b~ tr-n~fornln~ th~ ~cob-cteri-, and wher-by the mycobacteri- will ~pr--~ prot-ln(-) or polyp-ptidet9) \

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heterolo6ou~ to mycobacteria Such mycobacteria may be utllizedin the protuction of a vaccine or a therapeutlc a~ent, depend$ng upon the protein(s) or polypeptide9 e~pre~ed by the tran~formet mycobacteria To form ~uch a vaccine or therapeutic agent, the transformet mycobacteria arc administered in con~unction with a suitable pharmaceutical carrier As representative e~ample~ of suitable csrrier~ there may b~ mentioned mineral oil, alum, synthetic polymer~, etc Vehicle~ for ~accine~ ant therapeutic agent~ are well known in the art and the sel-ction of a suitable vehicle is d--med to bc within the ~cop~ of tho~- s~illed in the art from th- teaching~ contain~d herein Th- ~clection of a suitable vehicl- i~ al~o d~pendent upon th- manner in which the ~accine or therapeutic a8ent i~ to be admini~teret Th- vaccine or therapeutic a8ent m~ be in th~ for~ of an in~ectabl- ~o~e ant may be admini~teret intramu~cularly, intrav-nously, orally, intradermally, or by ~ubcutaneou~ a~minl~tration Other mean~ for ad~lniJtering th- ~accin- or therapeutic aBent should be apparent to tho~- ~killed in the art from the teaching~ hercin; accordingly, th- JCOp- of the in~e~tion i~ not to be limited to a particular d-livery form Wh~n th- tsansfor~-d mycobacteria are employed as a vaccine, such a vaccine haJ ipportant atvantag-~ over other pre~ently avsilsbl- vaccine- M~cobact-ria hav~, a~ hereinabov~ indicated, ad~u~ant properti-~ among th- b-~t curr-ntl~ known and, therefore, ~ti~ulat- r-cipient'~ immun- ~Jtem to r-~pond with 8r-at ff-cti~ n ~ Thi~ a~p-ct of th- ~accin- induce~
cell-~edi~t~d i_ unit~ and thu~ i~ eJpecially u~eful in providing immunit~ a~-in~t patho~-n~ in ca~-~ wher- c-ll-m-diat~d im~unity app--r- to b- critlcal for r~ tance ~l~o, m~cobact-rla m-y Jtimulat- lon~-t-r~ -mor~ or lmmunlt~ It thu~ ~a~ b- po~Jible to prim- lon~-la~tln~ T c-ll m-mor~, whlch ~tlmulateJ ~-condary antibot~ re~pon~ n-utrallzln~ to th~ in~ectiou~ ag~nt or the to~in Such prlmin~ of T c-ll m~ory i~ u~ful, for e~ample, , ~ r ~
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againJt tetanu~ and diphtheria to~in~ pertu~ malaria influenza viru~ Herpes ViN9, rabie~ Rift Valley fe~er viru~
tengue viru~ mea~le~ viru~ Human Immunodef~ciency Virus (HIV) respiratory syncytial viru~ human tumors and ~nake venoms Another atvantage in employing mycobacteria tran~formed with the phage DN~ portion of the pre~ent ~nvention as a vaccine or a therapeutic a8ent is that mycobaeteria in general haYe a large genome (i e appro~imately 3 ~ 106 ba~e pair~ in length) Because the genome i~ large it i~ abl~ to aeeommodAte a large amount of DN~ from other soure-(~) ant ~-y po~ibly be employed to make a vaccine and/or therapeutie a6ent cont~ining DNA
~equences eneoding more than on- anti8-n and/or therapeutic a6ent.
Al~o, becau~e th- inte~rated gen-(~) of intere~t ha~ not been lo~t following non-~-leeti~a growth o~ tran~formed myeobaeteria, th- gen-(J) of intere~t will eontinue to be e~pr-~sed by the tran~formet myeobaeteria following ad~ini~tration of th- ~ycobacteri- to a host Such ~yco~acteria therefore ar~ eff-eti~o ~ehiele~ for e~pre~sing antigen~ which stimulate an i N na re~pon~e or for th- e~pre~ion of th-rapeutie(l) a~-nt~ ~uch a- anti-tumor a~ents andlor antl-caneer a8-nt~
The in~ention wlll now be de~eribed with re~peet to the following ~u pl--; how-v-r th- ~eop- of the pre~ent in~ention i~ not to b- li~it~t th r-b~
- -- ,e~a~l- 1 Id-ntl~lcat~g~n oi th DN~ ~-qu-nce~ of th~ ttach~ent ~ite~
tt~. attL and ttR of M ~Y~C~ tl~

U~in~ ~t~nd~rd t-ehnolo~ , a lu~bd- EMBL3 llbr-r~ wa~
pr-par-d ~ro~ Ba H~ dl~-~t-d me261 ehro~o~o~al DN~ (~e261 i~ a ~tr-ln oi M~ ~n~ which lnelud-- n M ~o~ tl- ehro~o~om~
into which h-~ b--n intc~r~t-d th~ g~no~- o~ ~ycob-ctorial phage \

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, L5) and dig~ted with ~am HI Phag~ L5 contain~ DN~ ha~in8 restrietion ~it~s identical to tho~e of phag- Ll (Snapper et al 1988) e~eept that L5 i9 able to replicate at 42C and pha~e Ll i9 incapable of guch growth Thi~ library was then probed with a 6 7 kb DNA fragment isolated from the L5 genome that had been previously identifi~d as carrying the attP sequenc~ (Snapper et al 1988) One of the positive clone~ wa~ plaquo purified DNA
prepared and a 1 1 kb Sal I fragment (containing the AttL
~eq~ence) sub-eloned into sequencing vector pUC119 The DNA
sequence of this ~ra8ment wa~ determined u~ing a shotgun approach coupled with ~anger ~equçncing ~y isolating and ~equencing the attL ~unction ~it- and eomparing thi~ to the DN~ ~equ~nce of L5 that was availabl- a region was deter~ined wher~ th- two ~equ~nce~ align-d but with a ~peeifie di~eontinuity present The di~continuity r-present~ on- ~it~ of a core ~quenee whleh i~
identlcal in attP, attB and attL The r~gion eontaining the reeombinational ero~over point i9 ~hown in Fi~ur- 1 ~
Tha attL DNA (1 1 ~b Sal I fragment) wa~ u~-d a~ a probe to hybridize to a South-rn blot of Ba~ HI dige~ted me26 DN~ which is a strain of M ~-~ ati~ whieh inelude~ an M sme~matis chromo~ome without any phag- into~ration (Jaeob~ et al 1987 her-inabove eit-d ) ~ ~ingle band of appro~imately 6 4 kb wa~
deteeted eorre~pontin~ to th- attB ~equene- of M ~me~mati Thi~ ~am- attL prob- w-~ u~ed to ~ere~n co8nit library of me 6 (providet b~ Dr Plll Jaeob- of the ~lbert ~in~tein Colleg- of Medieine of Y-~h$~ Uni~-r~ity) and a nunb-r of po~iti~- cosmid elone~ w-r~ ld~ntlfi-d DN~ w-~ prepared fro~ the~e elone~ and a l i ~ 1 I fra8--nt (eontaining the attB ~it-) that hybridi~-~ to t~ attL prob- wa- ~ubelon~d into pUC119 for ~-qu-neln~ nt further n-ly~i~ Th- DN~ ~equ-ne- eontainin~ tho eor~ ~-qu-ne- w-~ deter~inad and i~ ~hown ln Fi~ur- 1 Th- eore ~equ-nee, whieh 1~ ldentleal ih attP attB and attL, ha~ ~ length of 43bp \

Thc mc261 la~bda EMBL3 library wa~ then prob~t with the 1 9kb Sall fragment containing the attB ~ite Po~iti~e plaque~
were identifi~d DNA wa~ prepared and analyzed by restriction analysi~ and Southern blot~ Lambda clone~ were itentified that contained a 3 2kb Bam HI fragment containing the putative attR
site The 3 2kb ~am HI fragment wa~ purified snd cloned into pUC119 for sequencing and furth-r analysi~
B DQtermination of attP-inteRrase rc~ion of L5 ~encme Concurrent with the above procedures a ~ignificant portion of the DNA sequenc~ of L5 had be-n dot~rminot and represented in ~everal conti~ or i~land~ of DN~ sequence SQquenc~ of the 6 7kb Bam HI frag~cnt h~r~inabovo de~cribed w-ro dQtermined by (a) analy~i~ of th~ location of Bam HI sitQ~ in the contigs of th~ DNA of L5 snd (b) by deter~ining a ~hort stretch of DNA
sequenco from around tha Bam HI ~it~ of plas~id pJR-l (Figure 6) wh~ch carrie~ th~ 6 7kb Bam HI frag~-nt of L5 A segm~nt of DN~ ~equ~nc~ wa~ locat~d that r~prosented the 6 7kb Bam HI fra8~-nt of phago L5 Studies of other phage~ have shown that th- integra~ ne~ are oft~n locat~d clo~e to the attP ~ite It W-J thu~ teten~ln~d that the LS inte~rasc (int) gene should lie either within the 6 7kb Ba~ HI fra8mQnt or in a DNA ~qu-nco on oith-r ~ide of it Tho DNA ~quence in the region~ wa~ tb~n analyz~d by tranJlating it into all 9i~ possible readin~ fram-~ and ~-arching th~o ad no ac~t ~equ-nces for ~imilarit~ to th- fa ily of intc8rase related protein9 and jthrough coo~uS~r-aJ~i~t~t analysis of tho DNA ~qu-nc~ As shown J in F~ur- 2, th r- ara ~hown ewO domain~ o~ r~a~onabl~ good con~rv-tion non~ LS int-gra~- and othar int-gr-~o~, and thrce amino acld r-~ldu-~ that ar- ab~olutaly con~orvad ln domain 2 (S~- Ya~ t al , J Mol Biol , Vol 207, P8~ 69S~717 tl9~9) and Poyart-Sal~-ron, ~t 1, J EMJO, VQ1. a, P~. 2425-2433 ~1989)). ~ r~lon wa- identi~l~t, and analy~i~ of the corr~sponding DNJ~ ~qu~rlc~ ~how~d a r~adin~ ~ra~ that could \

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encod~ for protein of appro~imately 333 amino aclds These observation~ identified the putatlve int gene The location of the int gene wa~ not withln the 6 7kb Bam HI
fragm~nt; however, it wa~ very close to it with one of thc ~am HI
sites (that define9 the 6 7kb Bam HI fra8ment) le~s than 100 bp upJtream of the start of the 8ene Analy~is of the Bam HI sites showet that the $nt gene lay within a 1 9~b ~am HI fragment located ad3acent to the 6 7kb ~am HI fragm-nt Thi~ l 9kb Bam HI
fragment wa~ cloned by purification of the fragment from a Bam HI
/ti~e~t of LS DN~ and clonin~ into pUC 119, to g-nerat~ pMHl ~/ (Figure 7) From a combinatlon of the above spproacho~, a Ich~matic of the organization of tho attP-int reglon of LS wa~ con~tructed J (Fi~ure 3), and the 8-nc ~equence of th- attP-int region i~ ~iven J in Fi~ur~ 4 C Con~truction of PMHS
Thc 6 7kb ~a~ HI fragMent of mycobacteriophage L5, which contain~ th- attP ~it-, a~ horeinabo~e d~Jcrlbed, wa~ cloned into J the Bam HI ~it- of pUC 119 (Figure 5~ This wa~ achieved by purifyin~ th~ 6 7kb Bu HI frag~ent from a Bam HI digest of L5 DN~ ~eparated b~ agaro~c 8~1 electrophorc~i~ ant ligatin~ with Bam HI cut pUC 119 DNA wa~ prcp-r~d fro~ candidat~ recombinants and charact~riz-d ~ rootriction onzym- an lysis and 8Cl ~l~ctrophor~ r~co~binant wa~ ldentified that contained the 6 7kb Ban HI-~r~g~-nt of LS clon-d into pUC 119 Thi9 plasm~d J wa~ na~ d pJR-l, a- hown in Fi~ure 6 Anal~-lo o~ DN~ J-qu-nco dat- fro~ a pro~ct eo ~2qu~nce L5 show-t th-t a 1 9~b Bu HI fra8~-nt at~acont to tho 6 7~b Bam HI
fragm~nt h-r-lnabo~ t-~crib-d cont-ln-d tho lnt~gra~a 8ona ~ pla~mlt containln~ l 9~b Ba~ HI fra~ont containing the DN~ encotinE ~or tho int-8ra~6 clon~d lnto th- Bam HI ~it~ of pUC
119 wa~ con~truct~d Th~ l 9~b frag~ont w~ purifict from 8 Bsm \

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J ` ~

HI di8e~t of L5 DN~ and cloned into the ~am HI ~ite of pUC 119 Construction of the recom~inant waJ determinet by re~triction analysis and ~el ~lectrophor-si~ Thi~ pla~mid wa~ called pMHl, the construction of which is shown schematically in Figure 7 pJR-l was then modifiet by dige9tion with EcoRI and SnaBI
(both are unique cloning site~, between which i~ a Bam HI site The Eco RI-Sna BI frag~ent, including the Bam H~ ~ite wa~
e~cised, and the plasmid wa~ religated to form plasmid of pMH2, which contains on- ~am HI site compared to two Bam HI ~ites contained in pJR-l A sche~atic of th- conJtruction of pMH2 i~
J shown in Figure 8 Th- 1 9k~ Bam HI fragm-nt, which includ~ the integrase gene, wa~ purified from a ~am HI dlge~t of pMHl and ligated to ~am HI digested pMH2 RecombinantJ were id~ntified aJ abo~a and the orientation of the l 9kb fragment determined ~ pla~mld called pMH4 wa~ thUJ con~tructed (Figur~ 9) in which th- region from th~ Sna BI sit- ~upJtream of attP) throu~h to th~ Bam HI
~ite (downstrea~ of the integraJ~ gene) wa~ identical to that in pMH4 w-J dige~ted with NindIII (unique ~ite) and was ligated to a lkb HindIII fra~ent purifie~ from p~D43 (~upplied by Keith Darby~hire of th- Ni~el Gindl~y Laboratory) that contains the gen- determining ro~i~tanc~ to kanumycin Recombinsnt~ were identified ant ch~ract~rized aJ above Thi~ plas~it i9 called J pMH5 ~ sche~atic o~ the con~truction of pMHS i~ shown in Fi~ure 10. ., D ~nt-aration of DMHS into attB of M ~m-~mati~
Pl~lt~ pYU~12 (- ~ift from Dr Blll Jacob~ a ~chomatlc of the for ation o~ whlch iJ ~hown in Fl~ur- 20), pMD01 (Fi~uro 11), ~~
and pMHS w r~ ctroporat-d, with four tl~foront concentratlon~
of plaJmld DN~ o~er a 1,000-fold ran8e, lnto M ~e~ ~tiJ ~traln mc21S5, a ~train which i~ sbi- to support pla~ld r~plicatlon IQ Exampl~- 1 and 2 all electroporation procedures of M
~me~mati~ or of BCG were carried out a~ follow~
Cultures of organism w-re grown ln Middlebroo~ 7H9 metia as de~cribed by Snapper et al (1988) harve~ted by centrifugation wa~hed three time~ wlth cold lOZ Klycerol and resu~pended at appro~$mately a 100 ~ concentration of cell~
1 ~1 of DNA wa~ added to 100 ul of cell~ ~n an ice- cold cuvette and pulsed in a Bio-Rad Gene Pulser and given a single pu19c at 1 25 kv at 2S ~F 1 ml of broth wa~ added the cells incubated for 1 hr at 37C for e~pre~ion of the antibiotic-resistant mark~r Cell~ w-re th-n concentrat~d and plated out on Middlebrook or tryptic ~oy media containin~ 15 ug/ml kanamycin Colonies were ob~erved after 3 to 5 days incubation at 37C
Each of pYUB12 pMD01 and pMHS carrie~ kanamycin resistance Pla~id pYUB12 carries an orlgin of DN~ roplication while pMD01 lac~ a m~cobact-rial origln of replication Pla~mid pMH5 doe~ not carr~ a m~cobacterial origin of r~plication but carries a 2kS reslon of phase LS whlch contain~ the attP site and the integra~- gcn- (Figure 4) The number of tranYformant~ were linear with DN~ conccneration Pl-~mid pYUB12 8i~e~ a lar~e number of tran~formant~ (2 ~ 105 pcr ~g DN~) in mc2155 while pMH5 give~ 6 ~ 10 tran~for~ant~ por ~ DN~ and pMD01 give~ no tran~for~-nt~
Th~ abo~ p-rl~-nt wa~ thon rep--tsd by ~lectroporating th pla~id~ p~U~12 pMD01 and pMHS lnto M ~m-~mati~ ~train mc 6 whlch to-~ not ~upport plaJmid ~ roplicatlon No tranifonn nta in c26 w-r- obtaln-t from pYU~12 or pMD01 whilo pMHS ~-v- ppro~ln-tel~ 104 ~ana~cln ro~i~t-nt tran~or~ant~ in mc26 p-r ~ of DNA, thu~ inticatln~ int-~ration of pMN5 into the mc 6 chromoso~-DN~ ~ro~ ~1~ intcp-ndcnt pMH5 tran~Yormant~ tfour in mc2 155 and two in ~c26) w-~ prepar~d The~e DNA ~ (along wlth DN~ fro~

both mc 15S it~elf, and mc 155 carrying the pla~mid pYU~12) were digested with a re~triction enzyme, and analyzet by Southern blot and hybridization with the M sme~matis 1 9kb attB probe hereinabove de~cribed As shown in Fl~ure 12, all 9i~
transformants have integratet into the attB site, resulting in the production of two new DN~ fragm-nts with diffcrent mobilities If pMH5 did not integrate into the attB site, it would be e~pected that a ~ingle ban~, corresponding to the attB
site in the mc2155 control, would be obtained E Construction of ~MH9 2 ant PMH9 4 pUCll9 wa~ dige~ted with HindIII, and a lkb HindIII
fragment, containing a kanamycin resistance ~cne, purified from p~D43, was li~ated to the HindIII dlgested pUC119 to form pMH8 J (Figur~ 13 ) ~ 2kb SalI fragm-nt of pMHS (bp 3226-5310~, which carries tho attP and integrase 8en- fro~ SalI di~sted pMH5, wa~
purified ant ins~rted in both orientatlon~ r~latlvo to th~ vector /backbon- of SalI ~igett~d pMH8 to form plasmit~ pMH9 2 and pMH9 4 J (Figure~ 14 and lS) F Stabilt~ of DlaJ~ldJ in M ~m-~matl~
_~k_~eY~ train mc2155 cell~ carrying, a~ a result of electroporation,~ pla~mid pYUB12, pMH9 2 or pMH9 4, or strain mc 6 cells carr~ing pla~id pMH5, a~ 8 re~ult of electroporation as her~inabove de~crlb-d, wer- grown to ~atura~ion in broth with kananmycin Cultur-~ w~ro th-n dlluted 1 100 into broth without kananycin and 4rown to saturation Two further cyclo~ of dilutlon snd- 4rowth w-r- don-, corr~pontlng to about 20 ~en ration- of b-ct-rlal growth Cult~r~ w~ro plated out to singl~ coloni-~ on non-~lectiv- plates, and appro~imately 100 of the~- colonl-J w-r- p-tch plat-d onto both non-~ol-ctiva and ~ cti~- pl-ta~ Sh- Z of coloni-~ th-t wor- ~on-itiva to kanaq cin, thu~ corro~ponting to tho p-rc-nt-~- of c~ which loJt th- pla~mld, 1J ~ivon b-row ln Tabl- I
Tabl~ I

. ' ,. -:

% lo~
pYUB12 (mc215S) 35 pMH5 (mc26) 17 pMH9 2 tmc2155) 3 pMH9 4 (mc2155) O
E~amPle 2 The 1 9 kb Sal I fragment, whlch includes the M 3me~matis attB site as hereinsbove de~cribed was clonet into pUCll9, and the plasmid generated wa~ named pMH^12 (Figure 16) ~-~ el purifi~d Sal I 1 9kb M sm ~-ti~ fragmont` containing attB (i~olated from pMH-12) was used to prob~ a South~rn transfer of Bam HI di8c~t-d mycobact~rial DNA'J, in~ludlng BCG substra~n Pasteur, ~hown in Figur~ 17 This demon~tratet that ther~ is one Bam HI fra~m~nt of BCG that strongl~ hybridizeJ to the M
sme~mati~ att~ prob~ and thr~ hybrldize w~akly The stronge~t hybridizing band i~ th- faJte~t ~o~ing band (appro~ioat-ly 1 9 kb) The ~am~ prob~ a~ abovo waJ uJ~d to prob- a BCG cosmid library (pro~id-d by Dr Blll Jacob-) and poJitiv- clone~ were id~ntified DN~ w~- pr-p-r-d from s-v~ral positi~ clones and analyzed by rc~triction analy~is and Southern blotting The 1 9 kb Ba~ HI fra~-nt (corre9pontin~ to tho strongly hybridiz~ng bant in thc South-rn blot) wa~ id~ntlfi-d, gel purifi-d from the cosmid DNA and clon~d into pUCll9 Tho ro~ulting pla~id wa~
na~ad pMH-15 (Pl~ur- 18) Pla~lda- pMH-S and pMH9 4 w~r~ ctroporat~d into BCG
Pa8t-ur It wa~ o~--rv-d thRt pMH~ 4 tran~form~ ~CG with high efflci~nc~ (-ppro~ t-l~ 104 tr~n~formant~/~g DN~), whll~ pMH-5 tr-n~fora- Bca t low fficl-nc~ 10 tr-n~onm-nt~/p~ ~N~) DN~ WaJ pr-p-r-d ~ron ~ca tr~n-~or~-nt~ nd ~n-ly~d b~ Bam HI
r-~trlctlon and South-rn blot n~ , probln~ with ~ol purifi~d l 9~b ~um HI BCG tt~ ~r~ ~nt ~ro~ pMH-15 $h~ dat~ ar~ ~hown in Fi~ur~ 19 ant Jhow th-t intogr~tion of Soth pMH S and pMH 9 4 \

~ -16-~ I ~ ,r r~

i~ sp~clPic to the BCG attB sita ti- the strongly cro~s-hybrldizing fraBment in BCG) This i~ illu~trated by the 109s of th~ 1 9kb Bam HI fragment from tho transformants and the appearanco of two new bant3 repre~enting attL and attR ~unction fragment~ Figure 19 shows ~u~t one of the pMH 5/8CG
transformant~, although all of the four that were analyzed show that one of the band~ (the large~t~ ls ~maller than e~pocted (and tifferent in each of the transformants) inticating that the transformation effiency of pMH 5 i9 low in BCG In contrast the four pMH 9 4 tran~formants ar- identlcàl to ~-ch other (Fi~ure 19) and give sttR and attL 3unct$on fragm-nt9 of the pretictet sizes.
E~-mPl~ 3 A Construction of Plasolts includln~ mYcobactorial ~romotor e~res~ion cass-tt-1 ConJtruction of ~YUB125 Plas~id pALS000, a plas~ld whlch contain~ an origin ofreplicatlon of M fortultu~ and described in Labidl et al FEMS Microbiol L-tt , Vol 30, p~s 221-22S (1985) and in Gene Vol 71, pgs 31S-321 (1988) i~ sub~-cted to a partial Sau 3A
digest and Skb frag~-nts are g-l purified ~ 5kb fragment i9 then lisat-d to Bu~ HI dlg-st-d pIJ666 (an E coli vcctor containlng an ~ coli ori~in of ropllcation and l~o carries neo~ycin-~ana~cin r-si~tanc~, a~ descrlbed in Ri~-r ot al Gene, Vol 65, p~ 83-91 (1988) to for~ plss~it pYUB12 A
sch-~atic of-th- forn-tion of plaJ~ld pYUB12 1~ shown in Fi~ure pYU~12 ~nd pIJ666 w-r- then transform-d into M ~meRmatis and BCC N-o ~cln~roslstant tran~form-nts th-t w-r- only obtain-t b~ pYUal2 tran~for~atlon conflr~ed th-t p~S000 conf-rr-d utononou- r-plicatlon to pIJ666 ln M ~ tis and BC¢
Shot~un muta~-n~ b~ Snappcr, ~t al (19~, hereinabo~o cited) inticat-d that no ~ore than half of the p~LS000 plasmid \

;

r- ~ J ~?

wa~ neceJ~-r~ to ~upport plasmid replication in BCG This segment pre~umably carried opcn reatins frame~ ORYl and ORF2, itentified by Rauzier, et al , Gene, Vol 71, pg~ 315-321 (9188), and al90 presumably carrlet a mycobacterial origin of replication pYU~12 i~ then dige~ted with ~paI and EcoRV, a 2586 bp casrying thi~ region or segment of p~L5000 i~ removed and ligated to PvuII dige~ted pYUB8 Pla~mld pYUB8 (a paR322 terivati~e) include~ an E coli replicon and a kanR (a~h) gene Ligation of the 2586 bp pYU812 fra8ment to PvuII digested pYUB8 re~ult~ in th- form-tion of pYUBS3, aJ t-pict-d in Figure 21 ~
Tran~formation of pYU~53 confirm-d that the EcoRV-HpaI fragment, de~ignatet M rep, wa~ capabl- of supportin~ autonomous replication in ~CG
Pla~mid pYUBS3 wa~ then digçJted with ~atI, EcoRV, and ~tI
in order to remov~ th- follow~ng re3triction ~it-J
~atI 5707 Eco~I S783 ~u~HI S791 SalI S797 P~tI S803 PJtI 72S2 SalI 725~
~a~HI 7264 EcoRI 7273 Cl-I 7298 - ~ HindIII 7304; and ~coRV 7460 Fr-~n nt end- re then flu-h-d wlth T4 DN~ pol~mer~e and ~ r-lig-t-d to for- pl-~nld pYUB125, con~tructlon o~ whlch iJ ~hown J ln Figur- 22 2 ~ in-tlon of ~uDer~luouJ vcctor DN~ fro DYUB12S
792 ba~e~ oi th~ tet geno, whch had been lnactl~ated by prior manipulatlon~, wa- eli in-t~d bg co~pl-t- N-rI dlge~t, \

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J ;~ 'J

gel purlfication of the 6407 bp fragm~nt, and ligation/recirculation, tran~formation of E coli strsin HB101, and ~election of KanR transformant~ The const N ction of the re~ultin~ plasmit, pMV101, i9 9chematically indlcated in Figure J 23, and the DN~ sequence of pMV101, which inclutes mar~in6s of reg$on~ which will be deleted and of mutations, a~ her~inafter te~cribed, is shown in Fi~ur~ 24 3 Elimination of unde~irabl~ r-~triction sites in aph (kan ~ ~ene To facilitat- future manipulatlon~, th- HindIII and ClaI
re~triction ~it-~ in th- aph g-n~ were mutagenized ~imultaneously by polymeras~ chain reaction PCR ~utag~ne~i~ accord~ng to the procedure d-~crib-d in Gene, Vol 77 PB~ S7-59 ~1989) The base~ chang~d in the aph g-ne wcr- at the third po~ition of codon~ (wobbl- ~a~e~) within each re~trlction ~lt- ant the ~ase subJtitution~ mat- w~r~ deJlgn~t not to chan~- th~ smino acid sequence of th- encodet prot-in Separate PCR reaction~ of pla~mit pMV101 with primers ClaMut-Kan ~ HindRMut-Kan and HindFMut-Kan ~ Bam-Kan were performet at 94C (1 ~in ), 50C (1 mln ), and 72C (1 ~in ) for 25 cycle~ The PCR primer~ had th- followinK ba~e sequences ClsMut-Kan CTT GT~ TG¢ O M GCC CC
HintRMut-K-n GTG AGA ~TG GC~ ~AA GAT TAT GC~ TTT CTT TCC AG
HlntFM~t~n GTC TC~ ~AA O~A ATG CAT ~T CTT TTG CCA TTC TCA CCG G
ca~ aa~ TCC J~C~ aa~ ca The r-~ultin~ PCR product~ w-re 8-1 purlflcd and ~i~ed and a ~in~le PC% r--ction without pri~-r~ w-~ p-r~or~-d t 94C (1 mln ), 72C (1 ~ln ) for 10 c~cle~ Prlmer~ ClaMut-Kan and ~a~-Kan wer~ add-d and PCR w-~ re~u~et at 94C (1 mln ), SOC (1 7 r~

min ), and 72C (2 min ) for 20 cycle~ The r-~ult~ng PCR
product ~Ran ~ut) wa~ di~e~ted wlth ~amHI and gel purifiet PlasmiLd pMV101 wa~ digested with ~laI and cohesive end~ were filled in by Rlenow ~ dCTP ~ dGTP Klenow wa~ heat inactivated and the digest waJ further digeJted with ~amHI The 5232 ba~e pair fragment wa~ gel purified and ml~ed with fragm~nt Kan mut and ligated The ligation was trsn~formed into E coli strain HB101 and Ran colonie~ were 9creened for plasmids re~i~tant to ClaI and HindIII digestion Such plasmid~ were designated a~
pMVllO, which i~ tepict~d in Figure 23 4 Elimination of ~auences not necessar~ for slasmid re~lication in m~cobacteria Plasmit pMVllO was resected in separate con~truction~ to yield pla~midJ pMYlll ant pMV112 In one con~truction, pMVllO
was di~ested w~th NarI and 8alI, th- endJ wer- filled in, and a S296 ba~e pair fra8ment waJ ligated and reclrcularizet to form pMVlll In anothor con~truct, pMVllO wu~ dige~ted with NdeI and SplI, the endJ were filled in, and a S763 ba~e pair fragment was li~ated and rocircul~rized to for~ pMN112 Schematic~ of the construction~ of pMVlll and pMV112 ar- Jhown in Figure 2S These construction~ furth r eliminatcd superfluouJ ~ coli vector sequences derived fro~ pAL5000 not noces~ary for mycobacterial replication Clonin~ wa~ perfor~ed in ~ coli Pla~mid~ pMVlll and pMV112 w ro t-~t~d ~or thc ability to replicat~ in M
~m-~msti~ B-cau~- both plas~idJ replicat~d in M sm-nm~ti~ the dele~ion8 of- ~ach pla~aid wcre combined to construct pMV113 To con~truct pMV113 (Figure 2S), pMVlll wa~ di~ested with BamH~ and ~coR~, and 1071 bp fragm-nt wa~ i~olated pMV112 wa~
di8-~t-t wlth Bu~HI nt EcoRI, and a 3~70 bp ~ragment wa~
iJolat-t, and th-n ligated to th- 1071 bp fr-g~-nt obtained from pMVlll to for~ pMV113 Th-~- con~truction~ thu- d-fined tha rcgion of pAL5000 n-c0~8ury for autonomou~ repllcatlon ln mycobact~ria a~ no l-r~r than 1910 ba~e pulr~
\

-~0-5 Mutai~en~ of re~triction ~iteJ in m,rcobacterial re~licon To faeilitat~ further manipulation~ of th~ mycobacterial replicon, PCR mutagenesi~ wa~ perforoed a~ above to eliminate the Sal I, EcoRI, and BglII site9 located in the open reading frame known as ORFl of p~LSOOO PCR muta~ene~is was perforn~ed at wobble ba9e~ within each re9triction ~ite and the ba e substitution~ w~r~ de~igned not to change the amino acid se~uence of the putative encoded ORFl prote~n The restriction sites were eliminated one at a time for te~tln~ in mycobacteria It wa~
po~ible to elininat- th~ SalI and EcoRI without altering replication in M 9me~mati~ In ono con~truetion, PCR
mutaRene~i~ wa~ perform-d at EeoRI1071 of pMV113 with primers Eco Mut - M rep and Bam-M rep to form pMV117, which laclcs the EeoRI1071 site Primer Eeo Mut - M rep ha~ the following sequenee TCC GTG C~ CG~ CGT GTG TCC CGG ~;
and }~ M rep h~ tho following ~-qu~nce C~C CCG TCC TGT aG~ TCC TCT AC
In another con~truetlon, PCR mutagen~ wa~ performed at the SalI 1389 ~lt- with primer Sal Mut - M rep ant ~am-M rep to form pMVll9, whieh laelu th- S-l~ 1389 ~ite Primer Sal Mut M rep ha~ th- following J-qu~nee TGG CG~ CCG C~G T'r~ CTC ~GG CCT
pMV117 wa~ th-n dlg-~ted wlth ~paLI and BglII, and a 3360 bp fragment w~ ol~t-d pMV119 w-~ dilpJt-d with ApaLI and BglII, and ~ 12~1 -b~r fra~-- nt wa~ i~olatad and ligatod to th- 3360 bp frag~-nt i~olat-d ~ros pMV117 to foml pMV123 ~ ~eh-~atie of the con~tructlon- of pl~nld- pMV117, pM~119, and pMV123 1~ shown in Figura 26 Fll in tlon of tha BglII Jita, how-v-r, ith-r by PCR
mu~a~-n-~i~ or Kl-now fill in, llminat-d pla~oid roplieation ln myeob~eteria, thu- u~ tinl~ that th~ ita i~ in pro~cimity to, or within ~aquane- n-eaJJar~r for myeob~eteria plasmid replieation \

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6 Con~truction of ~MV200 serie~ V~CtorJ
To ~-cilitat- manipulation~ of all th- component~ necesssry for plasoid replica~ion in E coll and mycobacteria, (E rep and M rep ) and s~lection of recombinants (KanR), ca~Qettes of each component were constructed for ~implifled a~sembly in future vectors and to lnclude a multiple cloning site (MCS) containing unique restriction site~ and tran~cription and tran~lation terminator~ Th- ca~ette~ wer- constructed to allow directional cloning and assembly into a plasmid where all transcription is unidirectional Kan Ca~tte A DN~ ca~-tte containing th- aph (KanR) 8~ne wa~
con~tructed by PCR with primer~ Kan5' and Kan3 An Sp~I site wa~ add-d to th- S' ent of PCR prlmer Kan3', re~ulting ~n the formation of a PCR prim-r havlng th- following ~equence CTC GAC TAG TGA GGT CTC CCT CGT G~A G
8a~ Hl ~ NheI ~it-~ were adt~d to th- 5' ~nt of PCR primer Kan5', reJultin~ in th for~atlon of a PCR prim-r having the followin~ ~equence PCR wa~ perforr-t at ba~e~ 337S and 458S of pMV123, and BamHI and Nh-I Jit-~ w-re add-t at ba~e 3159, ant an SpeI site waJ attet at baJ- 4585 Di8-~tion wlth BamNI ant SpeI, followed by purification r-~ult-d in ~ 1228/2443 KanR cas~-tt~ bounded by 8amHI and Sp-I coh ~ nd~ with th- dir~ction of tran~cription for th- aph ~an~ proc--ding fro~ Ba~NI to Sp- I
E r-~ c~ tta A D~A c--~-tt- cont~inin~ th- ColEI replicon of pUCl9 was con~tructot b~ PC~ with prim-r~ E r-p/Spe ant ~ rep/Mlu ~n SpeI
Jlt- w-- atd-d to th S' nd of PCR prlm-r ~ rep/Sp-, snd an MluI
~lt- wa- adt-d to tha S' nd o~ PCR prio-r ~ rep /Mlu Tho re~ultln~ prlo-r~ had th- followln~ ba~- ~equence~
~ r~ lS~
\

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CC~ CT~ GTT CC~ CTG AGC GTC AG~ CCC
E.reD./Mlu GAC M C GCG TTG CGC TCG GTC GTT CGG CTG.
PCR was performed at ba~-9 713 ant 1500 of pUCl9, ant an MluI site wa~ adted to bas~ 713, and a SpeI s$te wa~ added to base 1500 Digestion with MluI and SpeI, followed by purification resulted in an E rep cas~atte bounded by Spe~ and MluI cohesive end~ with the direct~on of tran~cription for RNA I
and RNA II replication primer~ proce-ding from SpeI to MluI
M reP cas~-ttc A DNA cas~ett- containing sequ~nce~ n-c~s~ary for plasmit replication in mycobactoria wa~ con~tructcd by PCR of pMV123 with prim~r~ M rep/Mlu and M r-p/Bam An MluI 3it- wa~ add-d to the S' end of PCa prim-r M rop/Mlu ~ BamH$ ~ite wa~ add-d to the 5' end of PCR pri~cr M r-p/B~m Th- r~ulting PCR primer~ had th-following ba~ ~equenc~
M re~ /Mlu CCA T~C GCG TGA GCC C~C C~G CTC CG
M reP /Bu~
CAC CCG TCC TGT GGA TCC TCT ~C
PCR wa~ pcrfor~cd at ba~ 134 and 2082 of pMV123 An MluI
sitc wa~ added to ba~o 2082 Dlg~tion with BamHI and MluI, follow-d by g-l pur~flc-tion re~ult~d in a 193S bs~e pair DN~
cass-tt- bount-d b~ MluI and Ba~H$ coha~lvo end~ with the direction of tran~cription for thc pALSOOO ORFl and ORF2 genes proc~-tin~ fr~ MluI to Bua HI
Th ~-n~, ~ rap, nd M rep PCR ca~ttc~ w~r- then mi~ed in eq~i~olar conc-ntration- and li~atet, and th~n tran~fonmed in . coll ~tr~in NB101 for J-loction of ~an tran~formant~
Colonio~ w-r- ~cr--n-t for th- pr-~-nc- of pla~mldJ c-~rying all thrc- ca~-tt~ t-r dig-ction wlth ~JHI ~ MluI ~ Sp-I and d~ n t~t pMV200 An dtltional rc~trlctlon ~lt-, NcoI, wa~
~liminat-d fro~ th M r-p c-~c~tt- by dig~tlon o~ pMV200 with \

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J

NcoI, fill ln with glenow, and li~ation and recircularization, resulting in the formation of pMV201 A schematic of the formation of pMV200 from pMV123 and pUCl9, and of pMV201 from JJ pMV200, i~ shown ln Figure 27 Pla9mid9 pMV200 and pMV201 were transformet into M sme~mati~ ant BCG Both plasmids yielded KanR transformations, thus indicatins their ability to replicate in mycobacteria J A synthetic multipl~ clonin6 ~equence (ffCS) (Figur~ 28) was then designed and synthesized to facllitate versatile molecular cloning and manipulation~ for foreign gen~ e~pressions in mycobacteria, and for integration into the mycobacterial chromosome The synth-tic MCS, shown in Figur- 28, contains 16 rostriction ~ite~ uniqu- to pMV201 and include~ a region carrying translation stop codon~ in eaeh of thr-- readin8 frame~, and a transcription t-nminator terlved fro~ E eoli 5S ribosomal RNA
(Tl) To in~ert th~ MCS ca~ette, pMV201 wa~ tise~ted with NarI
ant Nh~I, ant th- re~ulting fra8~-nt wa~ gel purifi-d The MCS
wa~ digestet with HinPI and Nh-I and, th~ resulting fragment was gel purifi~d Tho two frag~ent~ were then llg~ted to yield pMV204 A ~che~-tie of th- eon~truetion of pMV204 i~ shown in J Figure 29 Plasmid pMV204 w - th-n further manipulated to facilitate r-moval of th- M r-p ea~tto in further eon~tructlonJ pMV204 W8~ di~e~t~d with MluI, ~nt ~n MluI - Not I link~r W8~ insertet into th M~E ~lt- botw-en tho M r-p and th~ ~ rep to g~n~rate p~N206 ~ eh ~atie of th- eon~truetion of pMV~06 from pMVZ04 is C'~shown in Fl~ur- 30, ant th D~A sequence of pMV206 i~ ~iven in `~ Fi~ur- 31 7 Con~truetion of e~ora~ion ea~tt- b-~ed on ~CC HSP60 hmong th- ~o~t abuntant prot-ln~ in ~cobact-ria 1~ the HSP60 he-t ~hoc~ prot-ln (-l~o known a~ tho 65 kda antl~en) Beeau~ abundsneo of th- HSP60 prot-in in myeob-eteris indicates \

~t r~ ' f~
~trong HSP60 8~ne e~pre~ion th- s-quenc- contralling HSP60 e~pression wa~ choscn to control e~pre~9ion of heterologouJ genes encoding anti~ens or other proteins in BCG
The published ~equence of the BCG HSP60 gene (Thole et al Infect and Immun Vol 55 pgs 1466-1475 (June 1987)) and surrounting sequence permitted the con~truction of a cassette carrying e~pres~ion control 9equence9 (i ~ promoter and translation initiation sequences) by PCR Tha BCG HSP61 ca~ette (Figure 32) contains 375 base~ 5 to the BCG HSP60 start codon, ant 15 ~ase~ (5 codons) 3 to tha ~tart codon PCR
oligonucleotid- prim-r~ were then synthe~ized Prim-r Xba-HSP60 of th- following ~aqu-nc-C~G ~TC TAG ~CG GTG ACC ~C~ ACG CGC C
wa~ synthesized for th- 5 end of thc ca~-tt~ and primar Bam-HSP61 of th- followlng ~equenc-CT~ GGG ~TC CGC A~T TGT CTT GGC C~T TGwa~ synthe~izet for th- 3 end of th- ca~-tta Th- prim~r~ were u~ed to amplify th- ca~tt- by PC~ fro~ ~CG ~ub~train Pasteur chro~o~omal DNA Th- attition of th- Ba~ HI ~ita at th- 3 end of thc cas~ette atd~ on~ codon (A~p) to the fir~t si~ codons of th- HSP60 g-nc Each of pMV206 and th- PCR ca~ette HSP61 wa~ dige~ted with Xba I ant Bas HI Th- PCR ca~-tt- was thcn in~ert-d between the ~ba I ant Ba~ HI ~it-~ of pMV206, and th-n ligat-d to form pMV261, which iJ ~hown ln Figure 33 Th- E CD1i lac Z g-ne wa~ u~ed a~ a report-r, or mark~r gen- to a~ th abilit~ of the HSP61 c-~ett- to e~pre~s h-terolo~ou- ~-n - ln BCG ~ BunHI re~triction frag~ent c-rrying tb- l-c Z ~-n w~ clon-d into th- Ba~ HI ~lt- of ~a~ HI dlg-~ted pMV261, r-~ultlng ln th- forn tlon of pMV261/L2 ~ ~ch-~-tlc of th- con~tructlon af pMV261/L2 1~ ~hown ln Pl~ur- 34 Tho formatlon o~ pMV261/LZ ro~ultJ ln a fu~ion b~two-n tho H~P60 and lac Z g-n-~ at tha ~l~th codon of th- H~60 g-n- and tha ~i~th -~5-_ .

~J `~ . .f '`: 2 codon of tho l-c Z 6en- pMV261/LZ wa~ then tran~form~d into E
coli Jluo E coll colonies wero ~elected on ~-gal plate~ for the pre~ence of pMV261/LZ, ehus inticatlng that the HSP60 promoter and tran~lation initiation soquences were also acti~e in E coli pMV261/LZ waJ then tran~formed lnto BCG and plated on Dubofffl Oleic Agar plate~ containing ~-~al All BCG coloniè~ r-sulting from this transfonmation e~hibitcd blue color, thuq indicating that the lac Z gene product (a-galactosidase) wa~ e~pre~sed in BCG SDS polyacr~lamidc gel electrophore~is wa~ performed on ly~ate~ of the pMV261/LZ BCG recombinant~, revealing that B-galacto~ida~e protein waJ e~pre~9ed to level~ in e~ce~ of lOX
of total BCG protein (a~ det~rmined by ~tainin~ with Cooma~sie brilliant blue) The~- data indicated that BCG HSP61 e~pre~sion ca~tt- wa~ function-l in e~pre~Jlon v~ctor pMY261, and that ~ub~tantlal e~pre~Jion of a het-rologou~ gene could b~ achievet u~in~ HSP60 - controll-d expr-~ion in BCG
Plas~ld pMV261/LZ waJ then ~hown to replicate autonomously, and e~pre~ th- ~ coli B-galacto~lda~e, or lacZ g~n-, dr~ven by the BCG promotor HS~60, in M ~e~tlJ and BCG
B TranJfer of mYcobact-ri-l Pha~e L5 inte~ration ~ equence~ to ~CG e~Dre~ion ~-ctor PlaJ-it pMH9 4, wh~ch lnclude~ th- mycobact~rial phage L5 attP site and th- LS int-~ras- ~-ne, waJ dig-Jted to completion with either ~pnI ~ P w II or ~b-I ~ PvuII, and a re~triction fragment of 1862 or 1847 baJo pair~, re~pecti~ely, each of which contain th ttP ~it- and th- inte~ra~ gen~, wer~ purified by a~aros- ~ ctrophor~ Pl-~mid pMV261/LZ wa~ dige~ted wi~h ~baI ~ Dr-I to ~-n-r-t- ~ith r a 7S69 bp or 7574 bp vector fra~ment She 7S69 bp fra~ nt wa ted to the l862 bp fra~-nt d-rlvod ~ro~ pMH9 4 to for~ pMV460F/LZ Th- 7~74 bp fr~gm~nt W~ at-d to th- i847 bp ~ra6~-nt d-rlv-d fro~ pMH9 4 to ~orm pMN460 R/LZ Pla~id~ pMV460 F/L2 and pMV460R/LZ cach \

~;J`..' _ ". ., `- SJ
includ~ cobacterial replicon, th- L5 aetP ~it-, and the LS
integraJ- g~n- ~ ~ch-matic of th~ formation of pl-~midJ pMV460 F/LZ and pMV460R/LZ i~ ~hown in Figure 35 To generate derivati~e~ without the mycobacterial pla~mld replicon, pla~mids pMV460F/LZ and pMV460R/LZ were di~ested with NotI and recircularized b~ ation to generate pMV360F/LZ and pMV360R/LZ
~ schematic of the con~truction of pMV360F/LZ and pMV360R/LZ i9 ~hown in Figure 36 Plasmit~ pMH9 4, pMV261/LZ, pMV460F/LZ, pMV460R/LZ, pMV360F/L~, and pMV360R/LZ were then trantfonmed into M
~me~m~ti~ and ~CG to t-~t th~ir ability to r~plicat- autonomou~ly or integrate into the M Jme~mati~ or BCG chromoJome Tran~formatlon with pMH9 4, pMV261/LZ, pMV360F/LZ, and pMV360R/LZ
yielded kanan~ycin r--istant tranJform-nt~ of M JneRmat~ and 8CG Tran~for~ant~ of pMV261LZ, pMV360F/LZ, and pMV360R/LZ were shown to e~pr-JJ E coli B-~alacto~ld-~ b~ SDS-polyacr~lamide gel electrophore~iJ and ~-g-l a~ay a~ h-r-inabo~e d~cribed for pMV261/LZ Pla~nid~ pMV461F/LZ and pMV461R/LZ failed to yield kanamycin re~i~t-nt tr-nJfor~-nt~, thuJ indicating that chromo~om-l inte~r-tlon of a pla~mld carrying ~equ~nce~ mediating autonomou~ replicatlon i~ l-th-l to m~cobacteria E~a~ 4 A Con~tructlon o~ ~Pre~lon c-J~-tteJ baJ-d on BCG HSP70 ~ parti-l ~-qu-nce of the S' re~lon of tho ~CG HSP70 gene (whlch encot-~ for th BCG HSP70 h-at ~hoc~ prot-in, alJo known a~ th 70 ~d nti~-n) obt-in-d by Dr Rlc~ Young (MIT~
per~ltt-d th- con~tructlon of caJ~ett~J carryin~ e~preJ~ion control ~-qu-nc-r (1 - , pro~ot-r~ and tran~latlon initiation ~equenc-r) br PCR, ccortlng to th- proc~dure- h-r-in-bo~e c~ted Th- BCG-HSP71 c~r-tt- (~l~ur- 32) cont-in~ lS0 b--e- 5' to the ~CG-HSP70 ~t-rt codon nd lS b-~e~ (~ codon~) 3' eO the Jtart coton Prl~-r ~b--HSr70 W-J ~ynth-~lz~d for th- S' end o~ tho cas~etto, and prim-r Bam-HSP71 wa~ ~ynthe~izet ~or the 3' end of the ca~tte Th~ primers hat thc following ba~ quenc~
Xba-H5P~0 GGC CIC TAG ACC CGC ACG ACC AGC GTT ~GC
Bam-HSP71 GCT AGG ~TC CCC G~C CGC ACG ~GC C~T GGT
The primer9 were u~ed to amplify the ca~ette from BCG
substrain Pasteur chromosomal DNA The addition of the Bam HI
site at the 3' end of the cas~ette adts 1 codon (Asp) to the 3' ent of th~ HSP71 e~pro~ion ca~tt~
Each of pMV206 and the PCR ca9~ettc HSP71 wa~ tigo~tet with XbaI and BamHI The PCR ca~sette wa~ then in~ertet between the ~baI and ~a~HI ~it-~ of PMV 206, and then ligated to form pMV271, which i~ ~hown in Figure 33 B Clonin~ of H~V-lRac ~ BamHI-ClaI PCR ca~ette of H~V-l 8ag wa~ clon-d between th~ Bam HI and Cla I Jit~ of pMN261 and pMV271 to obtsin pMV261/gag and pMV271/g~g E~pr~c~ion of the ga~ anti6en~ in BCG
was ~erifi-d by the appearanc~ of immunoreactiv~ protein bandJ in WeJt-rn blot anal~ of ~ca pMV271/ga~ recombinant l~ateJ BCG
tran~formant~, how-v-r, wer- n~ver obtained with pMV261/gag, thus indicating thaS ga8 a~ e~pre~sed fro~ pMVZ6/gas wa~ lethal C Int-~ration of HSP60~ xDr-~lon ca~J-tte into BCG
In ord~r to t-Jt whoth~r int-gr~tion of an HSP-60-gag o~preg~ion C~J~-tt- into BCa wo~ld re~ult i~ non-l¢thal e~preJ~ion -0-~ ~a8 ln BCG, it wa~ decid~t that th~ HSP60-gag e~pre~ion c~ tto b- cloned into a plss~id (pMV307) which includ-8 th ~cobact-riophsnge LS attP and inte8ra~ sequ-nce~, tho con~truct~on of whlch i- e~pl-in-d h-reinbelow 1 ConJ~ruction o~ oMN307 Pla~it pMV206 wa- dl4-~tcd wlth NotI to r~ove the ~ycobact-rlal r-plicon The reJultlng 2209 bp frag~ent, which includc~ th- aph (RanR) gen-, the ~ coll rcplicon and th~

.

multlpl~ cloning ~itc wa~ ated and recircularized to form pMV205 tho con~truction of which i~ 9che~aticslly tepictet in Figure 30 PCR with primer~ XbaI-Att/Int and NheI-Att/Int was then performed on a S81 I fra8ment from pMH9 4 which contains the attP site and the L5 integra~e Bene The resulting ca~sette wa3 then dige~ted with ~baI and NheI and a 178~ bp fragment was gel purified pMV205 was then dige~ted with NheI and the resulting fragment was ligated to the 1989 bp fra8ment obtained from pMH9 4 to form pMV307 ~ ~chematic of th- con3truction of pMV307 is shown in Figurc 37 2 Con~truction and tran~formation of ~MV361/~a2 Th~ ~baI-ClaI HSP-antigen cass-tt- which includes th- HSP60 promot-r and HIV-lga~ ~-qu-nce~ W~J cloned b-twcen the NotI and ClsI ~it-~ of pMV307 to for~ pla~id pMV361/ga~ pMV361/gag was thon tran~for~-d into ~CG and Jhown to e~preJ~ HIV-lgag protein by W-stern blot analyJi~ with HIV-l infected hu--n ~era E~amDl- S
~ s-ri-~ of antigan 8-n- frag~ent~ or ca~ette~ were con~tructed b~ PCR with th- e~c-ption of th- 8-n- fragment containing th- g-n- for hu~an tumor antig~n p97 a~ indicated in Tabl~ 2 and clon-d in~to variou~ re~triction ~it-~ of pMV261 and pMV271 to for~ n-w con~truct~ of th- pMV261 ant pMV271 s-ries Th- antig-n ~-n-~ ~nti~-n 8-n- fra6 -nt~ clonin~ Jite~ u~et in pMV261 and pMV271 nd th- na~a~ of th- re~ulting construct~ are giv-n bolow-i~ T-bl- 2 -2~---- _ r 3n ~ ?J

O ._ ~, _ -- ~ E

, S S S S S S S S S _ ~ S C~ g e~ o O
Z ~ C, _ ~
C ~ y c c~ S ~ S S S S S S S S S S ~

~ ~E s s s ~ & ~ ^ ~ E

~ a a ~ O - ~ ~ ~ ~ ~

V -'~; ~ -~ - ~ z ~ s z 3 ~. ~ æ ~ ~ v -~ 0 -~ y eE
E ~, ~ ' ' ' _ _ V ~ _ ~ ~ ~ V V 1~ ~ V -- ~ ~
ur S E E S S SE S ~ _ SE ~ S S S 8 e ~ V I~ j E
e ~0 ~
c ,~ E

c ~ o` ~~ E
u E V ._ ~ o X æ ~ x _ ~ ,,~ 8 Ui a ~ O ~ c -- ~
c E, ~ g S S S S ~ S S S S S S c,~ Z

~ , ' Th~ followin~ con~truct~
pMV~61lP97 pMv271 pMV261/CS
pMV271/CS
pMV26 1/ SM97 pMV271/SM9~
pMV271lHIV- 1- 8a8 pMV26 l/HIV- lgp 120 pMV2 71 /HIV- lgp 120 pMV26 11HIV- 1- ~p4 1 pMV27 l/HIV- 1- 8P4 1 pMV261/HIV2ga~
pMV271/HIV2~ag pMV261/HIV2-~pl20 pMV271/HIV2-gpl20 pMV261/HIV2-gp41; ant pMV271/HIV2-~p41 wer~ tran~form-d into BCa, and tho pr~-ne~ of th- corresponding anti8en~ in BCG waJ verlfi~d by the appearanee of immunoreactiv-protein band~ in We~tern blot analy~i~ of BCG recombinant lysate~ , E~a Dle 6 Antigen 8-n- ~pr-~lon eaJ~ette~, whieh inelut- a promoter sequenee ant a h-t-rolo~ou~ gen- ~-qu-nee, wer~ ~ei~ed from the pMV261 ant pMN271 t-rl~ati~e~ wlth NotI and a ~-eond re~triction enzym ~it- (Pvu II, ~eo RI, Sal I, Cla I or Hind III) and clon~t into th- lnt-~ratln~ pla~mid pMV307 betwe~n th- NotI ~ite and a ~eond n~ lt- (Pvu II, ~co RI, Sal I, Cla I or Hind III) to form th- pMV 361~ and pMN371/~%~ ~-rl-~ o~ pla~mld~
pMV361/HIV-I~pl20) Tho b-ekbon-~ of tho~- ~orlo~ of pla~mid~
(pMV361 ant pMV371) ar- ~hown ln Flguro 38 '' Th- followln~ pl~ld~

.

pMV361/P97 pMV3~1/P97 pMV361/CS
pMV371/CS
pMV361/SM97 pMV371/SM97 pMV361/HlV-lga~
pMV371/HIV-lgag pMV361/HIV-lgpl20 pMV371/HIV-lgpl20 pMV361/HIV-lgp41 pMV3711HIV-lgp41 pMV361/HIV28 pMV371/HIV28~-pMV361/HIV2-gpl20; and pMV361/HIV2-SP4~
w~r- tranJformed into BCG and 3hown to e~pr-~ th- corresponting ant$g-n~ b~ W-~tern blot analy~l~ with the appropriate antig-n-specific hu~an ~-ra It i~ to be und-r~tood however that the scop- of the present in~-ntion iJ not to be limited to th- ~pecific emboti-ent~ de~crib-d abov- Th invention may b- practiced oth-r than a~ p-rticularl~ de~cribed and ~till b- within the scope of th- acco p-nying clai~a . . .

-3~-

Claims (11)

1. A DNA for integrating DNA into a mycobacterium chromosome, comprising:
a first DNA sequence which is a phage DNA portion encoding bacteriophage integration into a mycobacterium chromosome; and a second DNA sequence encoding a protein or polypeptide which is heterologous to the mycobacterium in which the DNA is to be integrated.

2. The DNA of Claim 1 wherein said phage DNA portion encodes mycobacteriophage integration into a mycobacterium chromosome.

3. The DNA of Claim 1 wherein said first DNA sequence includes DNA encoding integrase.

4. The DNA of Claim 3 wherein said first DNA sequence includes DNA encoding an AttP site.

5. The DNA of Claim 4 wherein said first DNA sequence encodes integration into BCG.

6. The DNA of Claim 5 wherein the DNA is a plasmid including an origin of replication for E. coli.

7. The DNA of Claim 1 wherein the first DNA sequence is selected from the group consisting of Figure 4 and analogues and derivatives thereof which encode for integration into mycobacteria.

8. Mycobacteria transformed with the DNA of Claim 1.

9. BCG having foreign DNA integrated into the chromosome.

10. A vaccine comprising: the transformed mycobacteria of Claim 8; and an acceptable pharamceutical carrier.

11. A process for providing a protein, comprising:
expressing the protein in the mycobacteria of Claim 8.