CA2110682A1 - Induction of ctl responses to foreign antigens expressed in mycobacteria - Google Patents

Abstract

A method of inducing a CTL response in an animal which comprises administering to the animal mycobacteria transformed with at least one DNA sequence which encodes a protein or peptide or fragment or derivative thereof which includes an epitope which is recognized by cytotoxic T lymphocytes. The mycobacteria are administered in an amount effective to induce a CTL response in an animal. In one embodiment, the transforming DNA sequence may encode an HIV
protein or fragment or derivative thereof.

Description

W~9~/2~76 PCT/~S92/04538 J ~ !~ 2 INDUCTION OF CTL RESPONSES TO FOREIGN
ANTIGENS EXPRESSED IN MXCOBACTERIA
This invention relates to the induction of a T-cell re~ronse, in particular a cytotoxic T l~mphocyte re~ponse. More p~rti~ularly, this invention relate~ to the induction of CTL
~e~r~nses to proteins or polypeptides expressed by recombinant mycohacteria.
Cell-mediated immunity (or CMI) of infe~tions is thouqht to ~e ~ major line of defense against ~ertain infections, such as ~ira] infections and certain bacterial infe~tions. For example, CMT m~y be significant in the development of an effective vaccine ~ n~t human immunodeficien~y virus (HIV), or AIDS virus, h~all~e HIV vaccines and/or therapies based on the generation of p~ive transfer of HIV-specific antibody in the absence of ~PIl-medi~ted immunity have not yielded consistent protection in pr~tes challenged with the HIV virus. Thus, interest has t.l~r?l~ to the induction of cell-mediated responses to various inf~tions, such as for example, HIV infection, and to the ~ tification of proteins or polypeptid~s that a stimulate a ¦ ~y~o~Qxic T lymphocyte response, and to methods of administering S~ proteins or polypeptides.
~ n accordance with an a~pect of the present invention, there p~ovided a method of inducing a CTL response in an animal ~o~ ing administering to the animal mycobacteria transformed witll at least one DNA sequence which encodes a protein or peptide ol~ ~r~gment or derivative thereof which includes an epitope which ` ~IIR~TITOTE SHET

W092/21376 rCT/US92/045~
71~0~8~ -2-i~ r~ognized by cytotoxic T lymphocytes. The mycobacteria are~minj.~tered in an amount effective to induce a CTL response in An ~nimal.
Proteins or polypeptides for which the at lea~t one DNA
: ~egllqn~e may encode, include, but are not limited to, : Myco~acterium lePrae antigens; Mvcobacterium tuberculosis An~.igells; Rickettsia antigens; ChlamYdia antigen~; Coxiella anti~en~; malaria sporozoite and merozoite proteins, such as the :~ ~ir~-lm~porozoite protein from Pla~modium berahei sporozoite~;
. Clo~tridium antigens; Leishmania antigens; Salmonella antigens;
Mycoh~cterium africanum antigens; MYcobacterium intracellulare ~- ~nt~gens; MYcobacterium avium antigens; E.coli antigens; Borrelia ntiqens; Li~teria antigens; Franci~cella antigens; Yersinia ~ntigen~ TrePonema antigens; Schistostoma antigens; Filaria t..iqen~; Pneumococcus antigens; StaphYlococcus antigens; Herpes .~ v~ ntigens; influenza and parainfluenza virus antigens;
9~ me~le~ viru~ antigens; mumps virus antigens; hepatitis virus ~ antiqens; Shiaella antigens; Bordatella antigens; Hemophilus i ~ antiqen~; Stre~tococcus ~ntigens; polio virus antigens; Rift I ~ ~
V~lley ~ever virus antigens; dengue virus antigens; Human Im~llno~ficiency Virus (HIV) antigens; and respiratory syncytial vinl~ (RSV) antigens.
In one embodiment, the at least one ~NA seguence encodes at t one protein or polypeptide or fragment or derivative thereo which includes an eptiope which is recognized by ytotoxic T lymphocytes induced by an HIV protein or fragment or deriv~tive thereof. The at least one DNA sequence may encode an HIV protein or fragment or derivative thereof. HIV proteins or polypeptides which may be encoded by the at least one DNA
~eq~ nce includes but are not limited to, HIV-I-gp 120; HIV-I-gp 41; HIV-I-gp 160; HIV-I-pol; HIV-I-nef; HIV-I-tat; HIV-I-rev;
HIV-I-vif; HIV-I-vpr; HIV-I-vpu; HIV-I-gag; HIV-2-gp 120;
HIV-2-gp 160; HIV-2-gp 41; HIV-2-gag; HIV-2-pol; HIV-2-nef;
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WOg2121376 2 1 1 0 ~ ~ 2 PCT/US92/04538 HIV-~-tat; HIV-2-rev; HIV-2-vif; HIV-2-vpr; HIV-2-vp~l; and HIV-?.-vpx.
Mycobacteria which may be transformed with the at least one DM~ ~sq~lence, which encodes a protein or polypeptide or fragment or ~rivative thereof which includes an epitope which is recogllized by cytotoxic T lymphocytes, include, but are not ljmit~d to, Mvcobacterium bovis-BCG, M.smeomatis, M.avium, M.phlei, M.fortiutum, M.lufu, M.Daratuberculosis, M.habana, M.scrofalaceum, and M.intracellulare. In a preferred embodiment, th~ mycobacterium is M.bovis-BCG or a mutant thereof.
The at least one DNA sequence may be contained within an e~pr~sion vector, which is transformed into a mycobacterium, whetehy the mycobacterium expresses the protein or polypeptide or fragment or derivative thereof which includes an epitope which is rec~gnized by cytotoxic T lymphocytes.
The expression vector may be, for example, a temperate h~lttle pha-mid or a bacterial-mycobacterial shuttle plasmid.
Each of these vectors may be used to introduce the at least one DNA sequence encoding a protein or polypeptide or fragment or derivative which includes an epitope which is recognized by cytotoxic T lymphocytes, stably into mycobacteria, in which the t ]e~t one DNA sequence may be expressed. When a shuttle ph~smid, which replicates as a plasmid in bacteria and a phage in `~` mycob~ctreia, is employed, integration of the phasmid, which incll~des the at least one DNA sequence encoding a protein or polyre~tide, or fragment or derivative thereof, which includes an ep~tnp~ which is recognized by cytotoxic T lymphocytes, into the mycohacterial chromosome occurs through site-specific int~ration. The at least one DNA sequence which encodes a prot~in or polypeptide or fragment or derivative thereof, which in~ es an epitope which is recognized by cytotoxic T lymp~locytes, is replicated as part of the chromosomal DNA. When t.erial-mycobacterial shuttle plasmid is employed, the at ` le~t. one DNA sequence which encodes a protein or polypeptide or '~
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WO g2r21376 PCr/US92/04538 2 1 1 J` 6 C~ 2 -4-fr~qment or derivative thereof, which includes an epitope whichis r~cognized by cytotoxic T lymphocytes, is stably maintained extr~chromo~omally in a plasmid. Expression of the at least one DN~ seqllence occurs extrachromosomally (e.g., episomally) For ex~mple, the at least one DNA seguence is cloned into a shuttle pl~mid and the pla~mid is introduced into a mycobacterium such aæ th~se hereinabove described, wherein the plasmid replicates epi~omally. Examples of such shuttle phasmids and bacterial-mycobacterial shuttle plasmids are further described in Application Serial No. 361,944, filed June 5, 1989, which is hereby incorporated by reference.
In one embodiment the mycobacteria are transformed with an expression vector which comprises at least one DNA sequence enco~ing a protein or polypeptide which includes an epitope which is rçcognized by cytotoxic T lymphocytes, and a promoter selected from the class consisting of mycobacterial promoters and mycobacteri~ophage promoters for controlling expression of the DNA
`~ encoding the heterologous protein or polypeptide, or fragment or derivative thereof, which includes an epitope which is recognized by cytotoxic T lymphocytes.
Mycobacterial and mycobacteriophage promoters which may be em~loy~d include, but are not lim~ted to, mycobacterial promoters uch as the BCG HSP60 and HSP70 promoters; the mycobactin promoter from M. tuberculosis and BCG; the mycobacterial 14 kda and 12 kda antigen promoters; the mycobacterial a-antigen promoter from M.tuberculosis or BCG; the MBP-70 promoter, the myc~bacterial 45 kda antigen promoter from M.tuberculosis or BCG;
the superoxide dismutase promoter; the mycobacterial asd promoter, and mycobacteriophage promoters such as the Bxbl, Ll, L5, and TM4 promoters. In one embodiment, the promoter is a mycobacterial heat shock protein promoter such as HSP60 or HSP70.
The promoter sequence may, in one embodiment, be part of an expression cassette which also includes a portion of the gene norm~lly under the control of the promoter. For example, when a :

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~:.: - , .. -WO92t21376 2 1 1 ~ ~ ~ 2 rcT/usg2/04s38 mycobacterial HSP60 or HSP70 promoter is employed, the expre~sion ca~sette may, within the scope of the present invention, include, in addition to the promoter, a portion of the gene for the HSP60 or HSP70 protein. When the expre~sion ca~sette and the DNA
encoding the protein or polypeptide, or fragment or derivative thereof, which includes an epitope which is recognized by cytotoxic T lymphocytes are expres~ed, the protein expressed by the cassette and the DNA encoding the protein or polypeptide is a fusion protein of a ragment of a mycobacterial protein (eg., the HSP60 or HSP70 protein), and the protein or polypeptide or fragment or deriviative thereof which includes an epitope which iæ recognized by cytotoxic T lymphocytes.
In a preferred embodiment, the transcription initiation site, the ribosomal binding ~ite, and the start codon, which provides for the initiation of the translation of mRNA, are each of my~obacterial origin. The stop codon, which stops translation of mRNA, thereby terminating 6ynthesis of the protein or polypeptide or fragment or derivative thereof which includes an epitope which i~ recognized by cytotoxic T lymphocytes, and the tr~n~cription termination ~ite, may be of mycobacterial origin, or o~ other bacterial origin, or ~uch stop codon and ~ n~ription termination site may be tho~e of the DNA encoding ;~ th~ pxotein or polypeptide which includes an epitope which is recogl1ized by cytotoxic T lymphocytes.
Preferably, the mycobacterial promoter is a BCG promoter, a11~ Q mycobacterium is BCG.
J1~ one embodiment, the expression vector may further include nM~ w1~ich encodes for proteins or polypeptides such as, but not lim;~d to, antigens, anti-tumor agents, enzymes, lymphokines, p~ m~cologic agents, immunopotentiators, reporter molecules of in~r~t in a diagnostic context, and selectable markers.
~ electable markers which may be encoded include, but are not l~mi~ to, the ~-galactosidase marker, the kanamycin resistance -~:
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m~r1c~r, the chloroamphenicol resistance mar~er, the neomycin resistance marker, and the hygromycin resistance marker.
In accordance with one embodiment, the vector further incl~1de~ a mycobacterial origin of replication.
In accordance with another embodiment, the vector may be a pla~mid. The pla~mid may be a non-shuttle pla~mid, or may be a fih~1ttle plasmid which further includes a bacterial origin of repli~ation such as an E.coli origin of replication, a Bacillus origin of replication, a StaDhYlococcus origin of replication, a Stre~tomvces origin of replication, or a pneumococcal origin of repli~ation. In one embodiment, the shuttle plasmid includes an E. coli origin of replication.
In accordance with yet another embodiment, the vector may .ftm~t~or include a multiple cloning site, and the DNA encoding for t)~ ~rotein or polypeptide, or fragment or derivative thereof, whi~11 includes an epitope which is recognized by cytotoxic T 1ymphocytes is inserted in the multiple cloning site.
In addition to the DNA oncoding a heterlogous protein or p~lyr~ptide, and the mycobacterial promoter for controlling ~xrre~ion of the DNA encoding the protein or polypeptide which in~ n epitope which is recognized by cytotoxic T ~ym~hocytes, the expression vector may, in one embodiment, 11r~ include a DNA sequenco encoding bacteriophage integration ~"t.~ ~ mycobacterium chromosome. Bacteriophages from which the DNA ~equence encoding bacteriophage integration into a m~ terium chromosome may be derived include, but are not limi~ to, mycobacteriophage~ such as but not limited to, the r.~, t.1, Bxbl, and TM4 mycobacteriophages; the lambda phage of E.
coli: the toxin pha~es of Corvnebacteria; phages of ActinomYcetes an~ Norcardia; the ~C31 phage of StreptomYces; and the P22 phage of Salmonella. Preferably, the DNA sequence encodes mycohacteriophage integration into a mycobacterium chromosome.
Th~ nMA ~equence which encodes bacteriophage integration into a mycoh~cterium chromosome may include DNA which encodes integrase, , ....
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W092/21376 2 1 1 ~ ~ ~ 2 PCT/US92/W538 whi~ is a protein that provides for integration of the vector int~ the mycobacterial chromosome. Preferably, the DNA ~eguence ~nc~it1g mycobacterial phage integration also includes DNA which e~1co~es an attP site.
The DNA encoding the attP site and the integrase provides for ~n integration event which i~ referred to as site-specific in~gration. DNA containing the attP site and the integrase gene iæ ~ap~ble of integrating into a corresponding attB site of a myco~Acterium chromosome ~ t is to be understood that the exact DNA ~eguence encoding the ~ttP site may vary among different phages, and that the exact D~1~ s~q~1ence encoding the attB site may vary among different mycobacteria.
Examples of expression vectors which include mycobacterial promoters and mycobacteriophage promoters, for controlling the at :
t one DNA sequence encoding a protein or polypeptide, or ; fragment or derivative thereof, which includes an epitope which recognizod by cytotoxic T lymphocyteq are further described in `~ application Serial No. 642,017, filed Jan~ary 16, l99l, which is a c~ntinuation of Application Serial No. 552,828, filed July 16, l9~0, now abandoned. The contents of Application Serial No.
642,017 Are hereby incorporated by reference.
In another embodiment, the mycobacteria are transformed with , ~ ~
DNA which comprises a first DNA sequence which is a phage DNA
portion encoding bacteriophage integration into a mycobacterium chr~mo.some, and DNA including the at least one DNA seguence anco~ g a protein or polypeptide, or fragment or derivative t11eteof, which includes an epitope which is recognized by cytotoxic T lymphocytes.
The term "phage DNA portion", as used herein, means that the DNA sequence is derived rom a phage and lacks the DNA which is reql1~red for phage replication.
Bacteriophages from which the phage DNA portion may be derived include, b~t are not limited to, mycobacteriophages, such :
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~ hl1t not limited to the tho~e hereinabove described.
Pr~f~rably, the phage DNA portion encodes mycobacteriophage int~ration into a mycobacterium chromosome.
In a preferred embodiment, the first DNA sequence includes DNA ~ncoding integrase, which is a protein that provides for integr~tion of the DNA into the mycobacterial chromosome. Most pre~rably, the first DNA sequence also includes DNA which encodes an AttP site.
The DNA sequence encoding the AttP site and the integrase rovid~s for an integration event which is referred to aæ
sit~-specific integration. DNA containing the AttP site and the int.~grase gene is capable of integration into a corresponding AttB site of a mycobacterium chromosome.
It is to be understood that the exact DNA seguence encoding the attP site may vary among different phages, and that the exact DNA ~equence encoding the attB site may vary among different mycobacteria.
The integration event results in the formation of two new junction sites called AttL and AttR, each of which contain part of *~ch o AttP and AttB. The inserted and integrated non-phage A w1-ich includes the first DNA sequence and the at least one N~ ~quence, which encodes a protein or polypeptide, or fragment or d~rivative thereo~, which includes an epitope which is recognized by cytotoxic T lymphocytes is flanked by the AttL and AttR sites. The insertion and integration of the phage DNA
portion results in the formation of a transformed mycobacterium.
~ he DNA may further include DNA which encodes a selectable m~rk~r or markers; or other proteins or polypeptides of interest, S~15h ~S, but not limited to anti-tumor agents, enzymes, lymrhokines, pharmacologic agents, immunopotentiators, and repo1^t.er molecules of interest in a diagnostic context.
~ electable markers which may be encoded ~nclude, but are not limited to, the kanamycin resistance marker, the neomycin SUBSTITUTE SHEET

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wo g2/2l376 ~ 1 10 ~ 8 2 PCT/US92/045~
_g_ re~tance marker, the chloroamphenicol resistance marker, and t}-~ hygromycin resistance marker.
The phage DNA portion of the present invention, which incl~ æ the first DNA sequence encoding mycobacterium phage int~gration into a mycobacterium chromosome, and the at least one DNA æequence encoding a protein or polypeptide, or fragment or deri.vative thereof, which includes an epitope recognized by cytotoxic T lymphocytes, may be constructed through genetic ~: engi.neering technigues known to those skilled in the art. In a preferred embodiment, the phage DNA portion may be a plasmid inclllding, in addition to the DNA encoding integration and the DNA encoding a protein or polypeptide, or fragment or derivative thereof, which includes an epitope recognized by cytotoxic T lymphocytes, an origin of replication for any of a wide variety o o~ganisms, which includes, but is not limited to, E.coli, StreptomYces species, Bacillus species, StaPh~lococcus species, Shigella ~pecies, Salmonella species and various species of pne~lmococci. Most preferably, the plasmid includes an origin of 1~ replication for E.coli.
The phage DNA portion also may include a suitable promoter.
it.~ble promoters include, but are not limited to, mycobacterial omot~rs and mycobacteriophage promoters such as those herein~bove described.
The promoter seguence may, in one embodiment, be part of an expression cassette which also includes a portion of the gene norm~ly under the control of the promoter, as hereinabove d~rihed. For example, when a mycobacterial HSP60 or HSP70 prom~t.el~ is employed, the expression cassette may include, in ~d~i~.i.on to the promoter, a portion of the gene for the HSP60 or T1~7n p~otein~ When the expression cassette and the DNA encodinq the protein or polypeptide, or fragment or derivative thereof, :~ wl~ incl~ldes an epitope which is recognized by cytotoxic T
lymr~ ytes are expres9ed, the protein expressed by the cassette ~n~ t.~ NA encoding the protein or polypeptide is a fusion ~'~

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W0~2~21376 PCT/US92/~538 21 1 0 6 82 -lo-protein of a fragment of a mycobacterial protein (eg., the HSP60 or HSP70 protein), and the protein or polypeptide, or fragment or derivaitve thereof, which includes an epitope which i~ recognized by cytotoxic T lymphocytes.
In a preferred embodiment, the transcription initiation site, the ribosomal binding site, and the start codon, which provides for the initiation of the translation of mRNA, are each of mycobacterial origin. The stop codon, which stops translation of mRNA, thereby terminating synthesis of the protein or polypeptide, or fragment or deriviatve thereof, which includes an ; epitope which is recognized by cytotoxic T lymphocytes, and the transcription termination site, may be of mycobacterial origin, or of other bacterial origin, or such stop codon and trat~cription termination site may be those of the DNA encoding the pr~tein or polypeptide, or fragment or derivative thereof, whic1~ includes an epitope which is recognized by cytotoxic `~ T Iymphocytes.
Examples of DNA which includes a first DNA seguence which is a ph~ge DNA portion encoding bacteriophage integration into a myco~terium chromosome, and DNA including the at least one DNA
ql~nce encoding a protein or polypeptide, or fragment or ri-~ative thereof, which includes an epitope which is recognized ~y ~ytotoxic T lymphocytes are further described in application fi~ri~l No. 553,907, filed July 16, 1990, the contents of which r~ hereby incorporated by reference~
M~cobacteria which are transformed which DNA which encodes f~t ~ protein or polypeptide or fragment(s) or derivative(s) t.l~ f, which includes an epitope which is recognized by c~t~t~xic T lymphocytes, may be employed in a composition, such ~s ~ ccine, for inducing a CTL response in an aniaml~ The v~ril-e may be administered to a human or non-human animal.
;~ T~ ~orm such a vaccine, the transformed myco~acteria are ~mi~listered in conjunction with a suitable pharmaceutical ~t ~ r. As representative examples of suitable carriers there " ~ `~

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W092~21376 PCT/USg2/04538 ulf~ 2 m~y ~ mentioned: mineral oil, alum, synthetic polymers, etc.
Vohi~les for vaccines are well known in the art and the selection ~f ~ s~litable vehicle i~ deemed to be within the scope of those ski)l~d in the art from the teachings contained herein. The so.)~tjon of a ~uitable vehicle is al~o dependent upon the manner in wl~ich the vaccine is to be administered. The vaccine may be in tl~e form of an injectable dose and may be administered intr~muscularly, intravenously, orally, intradermally, or by hcutaneous administration;
~ ther means for administering the vaccine or therapeutic q~nt. ~hould be apparent to those skilled in the art from the to~clings herein; accordingly, the scope of the invention is not to h~ limited to a particular delivery form.
he~l the transformed mycobacteria are employed as a vaccine, s~ vaccine has important advantages over other presently a~i 1 able. vaccines. Mycobacteria have, as hereinabove indicated, adjut.ant properties among the best currently known and, t~lot~ore, ~timulate a recipient's immune system to respond with re~t. effectlvenes~. This aspect of the vaccine induces ol l-m~iAted immunity and thus is especially useful in providing imm~ ity against pathogens in cases where cell-mediated immunity rr~ to be critical for resistance. Also, mycobacteria may t.im-ll ate long-term memory or immunity. It thus may be possible ^ r-imo long-lasting T cell memory, which stimulates secondary n~.~h~y responses neutralizing to the infectious agent. Such r~-iming of T cell memory is useful, for example, against r~ si~, malaria, influenza virus, Herpes virus, rabies, Rift Va~ fever virus; dengue virus, measles virus, Human Tmml1t~0~1eiciency Virus (HIV), and respiratory syncytial virus.
The invention will now be described with respect to the f~ willg examples; however, the scope of the present invention is ~t to be limited thereby.
ExamPle ,.,, '~
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W092~21376 PCT/US92/~S~
211~682 -12- `
. Construction of Plasmid includinq mYcobacterial ~romoter expression cassette and lacZ aene.
1. Construction of PYUB125 Plasmid pALSOOO, a plasmid which contains an origin of rerlic~tion of M. fortuitum, and described in Labidi, et al., FEMS Microbiol. Lett., Vol. 30, pgs. 221-225 (1985) and in Gene, V~l. 71, pgs. 315-321 (1988), is subjected to a partial Sau 3A
~ig~st, and 5kb fragments are gel purified. A Skb fragment is t.~on Iigated to Bam HI digested pIJ666 (an. E. coli vector ~nt~ining an E. coli origin of replication and also carries n~my~in-kanamycin resistance, as described in Kieser, et al., Gene, Vol. 65, pgs. 83-91 (1988) to form plasmid pYUB12. A
tic of the formation of plasmid pYUB12. A schematic of the f~m~.ion of plasmid pYUB12 is shown in Figure 1. pYUB12 and p~.~hh6 were then transformed into M. smeomatis and BCG.
M~emycin-resistant transformants that were only obtained by ~YUB1~ tra~sformation confirmed that pAL5000 conferred autonomous r~plication to pIJ666 in M. smeomatis and BCG.
Shotgun mutagenesis ~y Snapper, et al (1988, hereinabove ci~e~) indicated that no more than half of the pAL5000 plasmid w~ ceæsary to support plasmid replication in BCG. This ~ qm~nt presumably carried open reading frames ORFl and ORF2, i~ent~fied by Rauzier, et al., Gene, Vol. 71, pgs. 315-321 ~19~ nd also presumably carried a mycobacterial origin of repl.ication~ pYUB12 is then digested with HpaI and EcoRV, a 2586 ~r ~rrying this region or segment pAL5000 is removed and ligated to ~v~lII digested pYUB8. Plasmid pYUB8 (a pBR322 derivative) inc~ldes an E. coli replicon and a kanR (aph) gene. Ligation of the ~S86 bp pYUB12 fragment to PvuII digested pYUB8 results in thD formation of pYUB53, as depicted in Figure 2. Transformation of pYUBS3 confirmed that the EcoRV-HpaI fragment, designated M.re~, was capable of support~ng autonomous repl~cation in BCG.
Plasmid pYUBS3 was then digested with Aatl, EcoRV, and PstI
itl ot~er to remove the following restriction sites:

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W092/21376 PCT/USg2/04538 2 ~ 2 AatI 5707 EcoRI 5783 BamHI 5791 SalI 5797 PstI 5803 PstI 7252 SalI 7258 BamHI 7264 EcoRI 7273 ClaI 7298 HindIII 7304; and EcoRV 7460 Fragment ends are then flushed with T4 DNA polymerase and religated to form plasmid pYU8125, construction of which is shown in Figure 3.
2._ Elimination of suPerfluous vector DNA from PYUB125 792 ba-es of the tet gene, which had been inactivate~ by prior~manipulations, was eliminated by a complete NarI digest, gel~purl1cation of the 6407 bp fragment, and ation/recirculation, transformation of E. coli strain HB101, and ~lection of Kan transformants. The construction of r~ lting pla~mid, pMV101, is schematically indicated in Figure 4, ~nd the DNA ~e~uence of pMV101, which includes markings of regi~n~ whieh will be deleted, and of mutations, as hereinafter de~cribed, is shown in Figure 5.

3. ~on~truction of exPression cassette based on BCG HSP60.
~ ong the most abundant proteins in mycobacteria is the ~s~rl lleat shock protein (also known as the 65 kda antigen)~
8D~ e ~bundance of the RSP60 protein in mycobacteria indicates ~trol-~ HSP60 gene expression, the sequence controlling HSP60 exr~ ion was chosen to control expression of heterologous genes e~ ng antigens or other proteins in BCG.
The published sequence of the 8CG HSP60 gene (Thole, et al, Infect. and Immun., Vol. 55, pgs. 1466-147~ (June 1987)), and ::
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W092t21376 PCT/US92/04538 2110~82 -14-~-lrrollnding sequence permitted the ~onstruction of a cassette carrying expression control seguences (i.e., promoter and translation initiation sequences) by PCR. The BCG HSP61 ca~sette (Eiq-lre 6) contains 375 ba~es 5' to the BCG HSP60 start codon, and lS bases (5 codons) 3' to the start codon. PCR
oligonucleotide primers were then synthesized. Primer Xba-HSP60, of the following sequence:
CAG ATC TAG ACG GTG ACC ACA ACG CGC C
wa~ ~ynthesized for the 5' end of the cassette, and primer ~m-H~P6~, of the following sequence:
CTA GGG ATC CGC AAT TGT CTT GGC CAT TG
w~s ~ytlthesized for the 3' end of the cassette. The primers were u~ to amplify the cassette by PCR from BCG strain Pasteur c~r~m~omal DNA. The addition of the Bam HI site at the 3' end of tl)o cassette adds one codon (Asp) to the first six codons of the ~P60 gene.
Each of pMV101 and the PCR cassette HSP61 was digested with Nl)eJ and BamHI. The PCR cassette was then inserted between the ~n~ nd ~amHI sites of pMV101, then ligated to form plasmid ~MV6~ (Figure 7).
Tl~ E. coli lac Z gene (Figure 8) was u~ed as a reporter, or m~tl~^- qene to assay the ability of the HSP61 cassette to express h~ logous genes in BCG. A BamHI restriction fragment carrying ~.ho .l~c Z gene was cloned into the Bam HI site of Bam HI digested rMVfi~, reRulting in the formation of pMV65A/LZ as indicated ~hom~t.i~lly in Figure 7. The formation of pMV65A/LZ results in n between the HSP60 and lac Z genes at the sixth codon of tl~ r~ o gene and the sixth codon of the lac Z gene. pMV65A/LZ
w~ on transformed into E. coli. Blue E. coli colonies were s~ od on x-gal plates fo~ the presence of pMV65A/LZ, thus ~ t.ing that the HSP60 promoter and translation initiation S~-l~CS were also a~tive in E. coli.
pMV65A/LZ was then transformed into BCG and plated on Dubos ~loi~ Agar plates containing x-gal. All BCG colonies resultin~

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W092~21376 2 1 ~ O ~ ~ 2 PCT/US92/04538 fr~m this transformation exhibited blue color, thus indicating th~t. tl1e lac Z gene product (B-galactosidase) was expres~ed in RC~ DS polyacrylamide gel electrophoresis was performed on - lys~tes of the pMV65A/LZ BCG recombinants, revealinq that ~-ga1~ctosidace protein was expressed to levels in excess of 10%
~f t.~tal BCG protein (as determined by staining with Coomassie ~ril1i~nt blue). These data indicated that BCG HSP61 expression c~s~tte was functional in expression vector pMV65A.
ExamPle 2 Cytotoxic T lymphocYte resPonse to E. coli ~-galactosidase.
E. coli B-galactosidase was expressed in BCG as a six amino ~c~ f~sion protein with BCG hsp 60 protein using extr~c}1romosomal plasmid vector pMV65A/LZ utilizing the HSP60 prom~ter to drive expression. The recombinant BCG was grown to mJ~ Og phase in Dubos media and concentrated by centrifugation.
cteria were then re-~uspended in PBS plus 0.0~ Tween 80 ~:H~: An~ C~l~ sonicated brie.~ly to disperse clumped bacteria. Six week ld ~LB/c mice were inoculated with a single dose of 2 X lO2, 2 X 104, or 2 X 106 colony forming units (CFU'S determined p~t-inoculation) by either intradermal ~ID), intraperitoneal (IP), or intravenous (IV) injection. At 14 or l9 weeks ost.-.imm~nization splenocytes were harvested from mice and CTL
acti~ity was measured. CTL activity was measured as follows:
plenocytes (ACK-treated, 5 X lO6/ml) were stimulated in vitro in lO ml in upright T25 flasks by co-culture for 5 days wi~.h m.itomycin C-treated cells transfected with the lac Z gene (C~-4 cells; 5 X lO6/ml). A 4 hr. 5lCr release assay was then ~erformed in triplicate using P815 and Pl3.l cells (P815 cellæ
tr~n~fected with the lac Z gene) as targets. Various effector-target ratios were tested using 5,000 targets/well.
Specific lysis was calculated as follows: % specific lysis =
lO0 Y Irelease by effector cells minus spontaneous release/
m~xjmal release minus spontaneous releasel.

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WO92/21376 PCT/US92/~538 2110682 -16- """

~ t l9 weeks post-immunization, the remaining animals in each gr~ were boosted by intraperitoneal in;ection at lO l1g of p~1r.ified lac Z emulsified in incomplete Freund' 8 adjuvant (IFA).
Srl~ cytes were then harvested from these animals at 23 weeks And CTL activity wa~. again measured. Unimmunized ~nimal~ or animals immunized with lac Z emul~ified in IFA, or animals -~
imm~1nized with vaccinia virus expressing lac Z served as controls.
The results of the avove experiments, as determined by ~
~pecific lysis of target cells, indicated that a CTL response was in~lced in mice immunized with BCG transformed with the expr~ssion vector pMV65A/LZ.
ExamDle 3 Construction o inteqratina Dlasmid includina mYcobacterial promoter exPression cassette and HIV-I-qP 120 qene.
1. EliminatiQn of undesirable restriction sites in aPh (kanR) g,ene.
To facilitate future manipulations, the HindIII and ClaI
restri~tion sites in the aph gene in plasmid pMVlOl were mtlt~enized simultaneously by polymerase chain reaction (PCR) ta~eneæi~ according to the procedure described in Gene, Vol. 77 pq~. 57-59 (1989). The bases chanqed in the aph gene were at the th~r~ position of codons (wobble bases) within each restri~tion si.te ~nd the base substitutions made were designed not to change the ~mino acid sequence of the encoded protein.
~eparate PCR rea~tion~ of plasmid pMVlOl with primer C1~M~1t-Y~an ~ HindRMut-Kan and HindFMut-Kan + Bam-Kan were r~rf^1med at 90C (l min.), 50C (l min.), and 72C (l min.) for y~les. The PCR primers had the following base seqeuences:
ClaMut-Kan, CTT GTA TGG GAA GCC CC
HindRMut-Kan GTG AGA ATG GCA AAA GAT TAT GCA TTT CTT TCC AG
HindFMut-Kan SUBSTITUTE SHEET
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WO92/21376 2 1 ~ PCr/US92/04s38 GTG TGG AAA GAA ATG CAT AAT CTT TTG CCA TTC TCA CCG G
Bam-Kan CGT AGA GGA TCC AGA GGA CG
Tl~e res~llting PCR products were gel purified and mixed and a single PCR reaction without primers was performed at 94C (1 min.), 72C (1 min.) for 10 cycles. Primer~ ClaMut-Kan and Bam-Kan were added and PCR was resumed at 94C (1 min.), 50~C (1 min.), and 72C (2 min.) for 20 cylces. The resulting PCR
pro~lct (Kan. mut) was digested with BamHI and gel purified.
Pl~mid pMV101 was digested with ClaI and cohesive ends were filled in by Klenow ~ dCTP + dGTP. Klenow was heat inactivated Alld the di~est was further digested with BamHI. The 5232 base p~ir fr~gment was gel purified and mixed with fragment Kan.mut an~ lig~ted The ligation was transformed into E. coli strain 1 and KanR colonies were screened for plasmids resistant to Cl~J ~nd HindIII digestion. Such plasmids were designated as pMVllO, which is depicted in Figure 4.
2. _ Elimination of seauences not necessarY for Plasmid re~lication in mvcobacteria.
~l~smid pMVllO was resected in separate constructions to yiol~ rl~ids pMVlll and pMV112. In one construction, pMVllO
w~ liqeæted with NarI and BalI, the ends were filled in, and a ~o~ h~e pair fragment was ligated and recircularized to form rM~T~ In another construct, pMVllO was digested with NdeI and l I, ~9 ends were filled in, and a 5763 base pair fragment was l~a~ t~ and recircularized to form pMV112. Schematics of the t7 l-~tions of ~MVlll and pMV112 are shown in Figure 9. These ctions further eliminated superfluous E. coli vector ~ çes derived from pAL5000 not necessary for mycobacterial r~ tion. Cloning was performed in E. coli. Plasmids pMVlll r~lVll2 were tested ~or the ability to replicate in M.
sme~m~tis. Because both plasmids replicated in M. smeqmatis the d~ iolls of each plasmid were combined to construct pMV113.
(Fiqllre 9).

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2~106~2 -18-To construct pMV113, pMVlll was digested with BamHI and E~RI, and a 1071 bp fragment was isolated. pMV112 was digested with ~mHI and EcoRI, and a 3570 bp fragment was i~olated, and th~n ~i~ated to the 1071 bp fragment obtained from pMVlll to form rMV113. These constructions thus defined the region of pAL5000 n~c~ary for autonomous replication in mycobacteria as no larger th~n 1910 base pairs.
3.. Mutaqenesis of restriction site~ inLmycobacterial re~licon.
To f~cilitate further manipulations of the mycobacterial r~pli~on, PCR mutagenesis was performed as above to eliminate the ~ I, EcoRI, and BglII sites located in the open reading frame 3~n~m ~æ ORFl of pAL5000. PCR mutagenesis was performed at w~hhl~ hases within each restriction site and the base s~lh.~t~t~ltions were designed not to change the amino acid seguence o~ tl~o putative encoded ORFl protein. The restriction sites were elimin~ted one at a time for testing in ~ycobacteria. It was ~ p~ible to eliminate the SalI and EcoRI without altering `` ~ r~ tion in M. ~meomatis. In one construction PCR mutagenesis w~ r~r~ormed at EcoRI1071 of pMV113 with primers Eco Mut - M.rep ~n~ ~m-M.rep to for~ pMV117, which lacks the EcoRI1071 site.
Prim~ Eco Mut - M.rep has the following se~lence:
TCC GTG CAA CGA GTG TCC CGG A;
and Bam-M.rep has the following seguence:
CAC CCG TCC TGT GGA TCC TCT AC.
In another construction, PCR mutagenesis was performed at the ~nlI 1389 site with primer Sal Mut - M.rep and Bam-M.rep to orm ~MVll9, which lacks the SalI 1389 site. Primer Sal Mut-M.rep has the following sequence:
TGG CGA CCG CAG TTA CTC AGG CCT.
pMV117 was then digested with ApaLI and BglII, and a 3360 bp fr~gment was isolated. pMVll9 was digested with ApaLI and BglII, an~ A 1281 bp fragment was isolated and ligated to the 3360 bp fraqm~nt isolated from pMV117 to form pMV123. A schematic of the :

~ ~ SUBSTITUTE SHEET

211~5~2 -19- :
c~n~tr~lctions of plasmids pMV117, pMVll9, and pMV123 is shown in ~ig~lr~ 10. Elimination of the BqlII site, however, either by PCR
m~lt~enesis or Klenow fill in, eliminated plasmid replication in mycoh~cteria, thus æuggesting that the BglII site i8 in proximity to, or within a sequence necessary for mycobacteria plasmid replication.

4. Con~truction of PMV200 series vectors.
To facilitate manipulations of all the components necessary for ~lasmid replication in E. coli and mycobacteria, (E. rep. and M. r~p.) and selection of recombinants (KanR), cassettes of each comr~nent were constructed for simplified assembly in future vectors and to include a multiple cloning site (MCS) containing lniqlle restriction sites and transcription and translation terminators. The cassettes were constructed to allow directional cl~ning and assembly into a plasmid where all transcription is unidirectional.
KanR Cassette A DNA cassette containing the aph (KanR) gene was constructed by PCR with primers Kan5' and Kan3'. An SpeI site waæ added to the 5' end of the PCR primer Kan3', resulting in the for~tion of a PCR primer having the following sequence:
CTC GAC TAG TGA GGT CTG CCT CGT GAA G.
~ Bam HI ~ NheI sites were added to the 5' end of the primer ;~ KAn~', resulting in the formation of a PCR primer having the fol~owing sequence:
C~ AGG ATC CTT AGC TAG CCA CT GAC GTC GGG G.
P~R was performed at bases 3375 and 4585 of pMV123, and ~mt~T and NheI sites were added at base 3159, and an SpeI site w~ d~d at base 4585. Digestion with BamHI and SpeI, followed hy pl~rification resulted in a 1228/2443 Kan~ cassette bounded by ~U~ and SpeI cohesive ends with the direction of transcription for ~.h~ aph gene proceeding from BamHI to Spe I~
E~ reP cassette SUBSTITUTE SHEET

WO 92t21376 PC~/USg2/04538 h ~ 3 2 A DNA cassette containing the ColEI replicon of pUCl9 wa~ -~
con~tructed by PC~ with primers E.rep/Spe and E.rep/Mlu. An SpeI
site was added to the 5' end of PCR primer E.rep/Spe and an MluI
site was added to the S' end of PCR primer E.rep./Mlu. The res-llting primers had the following ~equenco~:
E reP./SPe CCA CTA GTT CCA CTG AGC GTC AGA CCC
E reP.~Mlu GAC AAC GCG TTG CGC TCG GTC GTT CGG CTG. -~CR was performed at bases 713 and 1500 of pUCl9, and an MluI site was added to base 713, and a SpeI site was added to b~se 1500. Digestion with MluI and SpeI, followed by pllrification resulted in an E.rep. cassette bounded by SpeI and M~al~ cohesive ends with the ~irection of transcription for RNA I
an~ RNA II replication primers proceeding from SpeI to MluI.
M.reP. ca~sette A DNA cassette containinq sequences necessary for plasmid replication in mycobacteria was constructed by PCR of pMV123 with ~rim~r~ M.rep/Mlu and M.rep/Bam. An MluI site was added to the 5' ~ of PCR primer M.rep/Mlu. A BamHI site was added to the 5' ~n~ ~- PCR primer M.rep/Bam. The resulting PCR primers had the :
foll~wing bàse ~e~lences:
M.rep.~Mlu ... . _ _ ~CA TAC GCG TGA GCC CAC CAG CTC CG
M.rep~ Bam C'~ CCG TCC TGT GGA TCC TCT AC
~ R was performed at bases 134 and 2082 of pMV123. An MluI
si t~l was added to base 2082. Digestion with BamHI and MluI, f~ wed by gel purification resulted in a 1935 base pair DNA
~ tt~ bounded by MluI and BamHI cohesive ends with the dir~ on of transcription for the pAL5000 ORFl and ORF2 genes rr~ from MluI to Bam HI.
The Kan , E.rep, and M.rep PCR cassettes were then mixed in e~llim~lar concentrations and ligated, and then trans~ormed in E.

SlJBS~l~U~E St~EE~

6 PCT/US92/04~
,,~ 21~0~2-21-.
coli ~train HB101 for selection of KanR tran~formants. Colonies w~r~ ~creened for the presence of plasmids carrying all three cAæ~sttes after digestion with BamHI + MluI ~ SpeI and designated pMV~.~O. An additional restriction site, NcoI, was eliminated from ~he M.rep cassette by digestion of pMV200 with NcoI, fill in wi~h r~lenow~ and ligation and recircularization, resulting in the for~tion of pMV201. A schematic of the formation of pMV200 from ~MVl~.~ and pUCl9, and of pMV201 from pMV200, is shown in Figure lJ. Pla~mids pMV200 and pMV201 were transformed into M.
~me~m~tis and BCG. Both plasmids yielded KanR transformants, t,hll~ indicating their ability to replicate in mycobacteria.
~ ~ynthetic multiple cloning sequence (MCS) (Figure 12) was th~tl ~esigned and synthesized to facilitate versatile molecular clol-ing and manipulations for foreign gene expressions in my~h~cteria, and for integration into the mycobacterial chromosome. The synthetic MCS, shown in Figure 12, contains 16 r~t.riction sites unique to pMV201 and includes a region carrying tr~n~lation stop codons in each of three reading frames, and a tr~n~cription terminator derivod from E. coli 5S ribosomal RNA
(Tl).
To insert the MCS cassette, pMV201 was digested with NarI
All~ Ml~eI, and the resulting fragment was gel purified. The MCS
w~s ~ig~sted with HinPI and NheI and, the resulting fragment was 1 r~lrified. The two fragments were then ligated to yield pMV?.~4. A schematic of the construction of pMV204 is shown in Fiq~lr~ 13.
~ la~mid pMV204 was then further manipulated to facilitate remov~l. of the M.rep cassette in further constructions. pMV204 was digested with MluI, and an MluI - Not I linker was inserted into the MluI site between the M.rep and the E.rep to generate pMV206. A schematic of the construction of pMV206 from pMV204 is sho~n in Figure 14, and the DNA sequence of pMV206 is given in Fig~lre 15.
5. Construction of exDre~sion cassette based on BCG HSP60.

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211~682 -22- " ~ ;

The HSP61 cassette (Figure 6) was constructed as hereinabove described in Example 1.
Each of pMV206 and the PCR cassette HSP61 was digested with XbaI and BamHI. The PCR cassette was then inserted between the Xb~I and BamHI sites of pMV206, then ligated to form plasmid pMV261. The construction of this plasmid is shown schematically in Figure 17. The reading frame and the restriction sites of the -`
multiple cloning site of pMV261 is shown in Figure 16.
The E. coli lac Z gene was used as a reporter, or marker gene to assay the ability of the HSP61 cassette to express heterologous genes in BCG. A BamHI restriction fragment carrying th~ Z gene was cloned into the Bam HI site of Bam HI digested pMV~61, resulting in the formation of pMV261/LZ. A schematic of th~ construction of pMV261/~Z is shown in Figure 18. The foJm~tion of pMV261/LZ results in a fusion between the HSP60 and lac Z genes at the sixth codon of the HSP60 gene and the sixth codon of the lac Z gene. pMV261/LZ was then transformed into E.
coli. Blue E. coli colonies were selected on x-gal plates for the ~esence of pMV261/LZ, thus indicating that the HSP60 pr~m~t~r and translation initiation sequences were also active in E. coli.
M~r~61/LZ was then transformed into BCG and plated on Dubos Ol~ gar plates containing x-gal. All BCG colonies resulting fl'~m t~liS transformation exhibited blue color, thus indicating tl-~ the lac Z gene product (B-galactosidase~ was expressed in ns polyacrylamide gel electrophoresis was performed on ~ t~ of the pMV261/LZ BCG recobinants, revealing that B-a~l~ctosidase protein was expressed to levels in excess of 10%
~f t~t~l BCG protein (as determined by staining with Coomassie hri ] 1 i ~nt blue~. These data indicated that BCG HSP61 expression te was functional in expression vector pMV261.
Plasmid pMV261/LZ was then shown to replicate autonomously, ~ rpresæ the E coli B-galactosidase, or lacZ gene, driven by th~ ~C~ promoter HSP60, in M. smeamatis and BCG.

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. . .

6. Transfer of mvcobacteriophaae L5 intearation sequences to BCG ex~ression vector.
Plasmid pNH9.4, which includes the mycobacteriophage L5 attP
~ite, And the L5 integrase gene, was employed in providing the L5 int~gration sequences to a BCG expression vector. The construction of pMH9.4, as well as it~ integration into M.
~megmatis and BCG, i~ de~cribed below in sections (i) through (vi ) ~
(i) Id_ntification of the_DNA sequences of the attachment sites, attB, attL, and attR, of M.smeamatis~

Uæing standard technologies, a lambda EMBL3 library was constructed u~ing chromosomal DNA prepared from mc261 (a strain of M. sm~gmatis which includes an M. smeqmatis chromosome into whicll has been integrated the genome of mycobacterial phage L5) ~nd digested with Bam HI. Phage L5 contains DNA having rest~iction sites identical to those of phage Ll (Snapper, et al.
1988), except that L5 is able to replicate at 42C and phage Ll is incApable of such growth. This library was then probed with a 6.7 kb DNA fragment isolated from the L5 genome that had been previollsly identified as carrying the attP ~equence (Snapper, et al 1988). One of the positive clones was plaque purified, DNA
prepared, and a 1.1 kb Sal I fragment (containing the AttL
seq~lence) sub-cloned into sequencing vector pUCll9. The DNA
seq~ence of this fragment was determined using a shotgun approach co~l~led with Sanger sequencing. By isolating and sequencing the attL, junction site and comparing this to the DNA sequence of L5 th~t was available, a region was determined where the two seq~lences aligned but with a specific discontinuity present. The discontinuity represents one side of a core sequence, which is identical in AttP, attB, and attL. The region containing the recombinational crossover point is ~hown in Figure 19.
The attL DNA (1.1 kb Sal I fragment) was used as a probe to hybri dize to a Southern blot of Bam HI digested mc26 DNA, which SUBS~ITUTE SHEET

W092/21376 PCT/US92/~538 ~ 1 1 0 ~ 8 ~ . ~! q -~4-i~ ~ strain of M. fimeqmatis which includes an M. ~meomatis ~hr~osome without any phage integration (Jacobs, et al, 1987, her~inabove cited.). A single band of approximately 6.4 kb waæ
~et~cted corresponding to the attB sequence of M. smegmati 8 .
Thi~ ~ame attL probe was used to screen a co~mid library of mc26 (provided by Dr. Bill Jacobs of the Albert Einstein College of Medicine of Ye~hiva University), and a number of positive cosmid clones were identified. DNA waæ prepared from these clones, and a 1.9 kb Sal I fragment (containing the attB site) that hybridizes to the attL probe was ~ubcloned into pUC119 for ~eq~encing and further analysis. The DNA sequence containing the core seqllence was determined and is shown in Figure 19. The core seq~len~e, which is identical in attP, attB and attL, has a length of 43bp.
The mc261 lambda EMBL3 library was then probed with the 1.9kb SalI fragment containing the attB site. Positive plaques were identified, DNA was prepared, and analyzed by restriction an~lysis and Southern blots. Lambda clones were identified that contained a 3.2kb Bam HI fragment containing the putative attR
sit~. The 3.2kb Bam HI fraqment was purified and cloned into ptJCl]9 for sequencing and further analysis.
(ii) Determination of attP-intearase reqion of L5 qenome.
Concurrent with the above procedures, a significant portion of the DNA sequence of L5 had been determined and represented in several "contigs" or islands of DNA sequence.
Seq~lences of the 6.7kb Bam HI fragment hereinabove described were det~rmined by (a) analysis of the location of Bam HI sites in the contigs of the DNA of L5, and (b) by determining a short stretch of ~ sequence from around the Bam HI sites of plasmid pJR-l (Fig~re 24), which carries the 6.7kb Bam HI fragment of L5.
~ segment of DNA sequence was located that represented the 6.7kh Bam HI fragment of phage L5. Studies of other phaqes have showll that the integrase genes are often located close to the at~.~ site. It was thus determined that the L5 inteqrase (int) SUBSTITUTE SHEET

WO 92t21376 2 1 1 0 ~j ~ 2 PCl`/US92/04~38 . . .

gen~ should lie either within the 6.7kb Bam HI fragment or in a DM~ ~equence on either side of it. The DNA sequence in the r~gions was then analyzed by translating it into all six possible re~in~ frames and searching these amino acid sequences for simi]arity to the family of integrase related proteins, and thro~lgll computer-assisted analysis of the DNA ~equence. As shown in Figure 20, there are shown two domain~ of reasonably good co~rvation among L5 integrase and other integrases, and three ~min~ acid residues that are absolutely conserved in domain 2.
(See Yagil, et al., J. Mol. Biol., Vol. 207, pgs. 695-717 (1989), and royart-Salmeron, et al., J. EMB0., Vol. 8, pgs. 2425-2433 (3q~a)). A region was identified, and analysis of the corr~sponding DNA ~equence ~howed a reading frame that could enca~e for a protein of approximately 333 amino acids. These o~elvations identified the putative int gene.
~` The location of the int gene was not within the 6.7kb Bam HI
fr~gment; however, it was very close to it with one of the Bam HI
site~ (that defines the 6.7kb Bam HI fragment) less than 100 bp upstream of the start of the gene. Analys~s of the Bam HI sites ~howe~ that the int gene lay within a 1.9kb Bam HI fragment ]~c~ted adjacent to the 6.7kb Bam HI fragment. This l.9kb Bam HI
fr~gment was cloned by purification of the fragment from a Bam HI
digest of L5 DNA and cloning into pUC 119, to generate pMH1 (Fig~lre 25).
From a combination of the above approaches, a schematic of the ~rganization of the attP-int region of L5 was constructed (Figllre 26), and the gene sequence of the attP-int region is gi~en in Figure 22.

(iii~ Construction of ~MH5.
The 6.7kb Bam HI fragment of mycobacteriophage L~, which contains the attP site, as hereinabove described, was ~loned into the Bam HI site of pUC 119 (Figure 23). This was achieved by purifying the 6.7kb Bam UI_fragment from a Bam HI digest of L5 " ` .

SU~Sl-ITUlE

2 11 0 6~ 2 -26- 5 DNA ~eparated by agarose gel electrophoresi~, and ligating with B~m ~ cut pUC ll9. DNA was prepared from candidate recombinants, and characterized by restriction enzyme analysis and gel a~ trophore,sis. A recombinant was identified that contained the 6.7kh Bam HI fra~gment of L5 cloned into pUC ll9. This plasmid w~s 1~Amed pJR-l, as shown in Figure 24.
Analysis of DNA se~uence data from a project to sequence L5 ~,how~d that a l.9kb Bam HI fragment ad;acent to the 6.7kb Bam HI
fr~gme11t hereinabove described contained the integrase gene.
A plasmid containing a 1.9kb Bam HI fragment containing the DMA ~nncoding for the integrase cloned into the Bam HI site of pUC
119 was constructed. The l.9kb fragment was purified from a Bam est of L5 DNA and cloned into the Bam HI site of pUC ll9.
~onstr~1ction of the recombinant was determined by restriction ~n~1y~is and gel electrophoresis. This plasmid was called pMHl, the ~onstruction of which is shown schematically in Figure 25.
pJR-l was then modified by digestinn with EcoRI and SnaBI
(~oth are unique cloning "ites), between which is a Bam HI site.
The ~coRI-SnaBI fragment, including the Bam HI site was excised, And the plasmid was religated to form plasmid of pMH2, which ' ~ont~,in~, on Bam HI site compared to two Bam HI sites contained in ~i.lP- ~ . A schematic of the construction of pMH2 is shown in Fi~1re 26.
~ he l.9kb 8am HI fragment, which includes the integrase g~n~, was purified from a Bam HI digest of pMHl and ligated to B~m HI digested pMH2. Recombinants were identified as above and tl1~ nrientation of the l.9kb fragment determined. A plasmid c~ d pMH4 was thus constructed (Figure 27) in which the region from the Sna BI site (upstream of attP) through to the Bam HI
site (downstream of the integrase gene) was identical to that in L5.
pMH4 was digested with HindIII (unique site) and was ligated to ~, lkb HindIII fragment purified from pKD43 (supplied by Keith Darbys}-ire of the Nigel Gindley Laboratory) that contains the SUBSTITUTE SHEET

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W092t21376 2 1 l O G ~ 2 PCT/USg2/045~

gen~ determining resistance to kanamycin. Recombinants were identified and characterized as above. This plasmid is called pMH~. A schematic of the construction of pMH5 i~ shown in Figure 28.
(iv) Inte~ration of DMH5 into attB of M. smeqntatis.
Plasmids pYUB12 ~a gift from Dr. Bill Jacobs, a schematic of th~ formation of which is shown in Figure 1), pMD01 (Figure 29), ~nd pMH5 were electroporated, with our different concentrations of rl~mid DNA over a 1,000-fold range, into M. smeamatis strain m~2]~, a strain which is able to support plasmid replication.
In ~ections (iv) through (vi), all electroporation procedures of M. fimegmatis, or of BCG, were carried out as follows:
Ctlltures of organism were grown in Middlebrook 7H9 media, as ~e~libed by Snapper, et al. (1988), harvested by centrifugation, w~h~d three times with cold 10% glycerol, and resuspended at proxim~tely a 100 x concentration of cells.
1 of DNA was added to 100 ~1 of cells in an ice-cold i v~tte and pulsed in a Bio-Rad Gene Pulser, and given a single p~ at 1.25 kv at 25 ~F. 1 ml of broth was added the cells in~ ted for 1 hr. at 37C for expression of the antibiotic-t~nt marker. Cells were then concentrated and plated out onMi~llebroQk or tryptic soy media containing 15 ~g/ml kanamycin.
Col~ es were observed after 3 to 5 days incubation at 37C.
Æ~ch of pYUB12, pMD01, and pMH5 carries kanamycin re~ nce. Pla~mid pYUB12 carries an origin of DNA replication, wl~ MDOl lacks a mycobacterial origin of replication. Plasmid p~r~ does not carry a mycobacterial origin of replication, but c~rt~ a 2kb region of phage L5 which contains the attP site and t~ tegrase gene (Figure 22). The number of transformants were lin~r with DNA concentration. Plasmid pYUB12 gives a large nt~m~er of transformants ~2 x 105 per ~g DNA) in mc2155, while rM~ gives 6 x 104 transformants per ~g DNA, and pMD01 gives no tran~formants.

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WOg2/213~6 PCT/US92/~S38 211~2 -28- "

The above experiment was then repeated by electroporating th~ plasmids pYUB12, pMD01, and pMH5 into M. smeomatis strain mc 6, which does not support pla~mid replication. No tr~n~formants in mc26 were obtained from pYUB12 or pMD01, while pMH5 gave approximately 104 kanamycin resistant transformants in mc 6 per ~g of DNA, thus indicating integration of pMH5 into the mc26 chromosome~
DNA from six independent pMH5 transformants (four in mc2 155 and two in mc26) was prepared. These DNA's (along with DNA from both mc2155 itself, and mc2155 carrying the plasmid pYUB12) were dig~ted with a reætriction enzyme, and analyzed by Southern blot and hybridization with the M. smeqmatis l.9kb attB probe hereinabove described. As shown in Figure 30, all six transformants have integrated into the attB site, resulting in the production of two new DNA fragments with different mobilities. If pMH5 did not integrate into the attB site, it wo~lld be expected that a single band, corresponding to the attB
site in the mc 155 control, would be obtained.
(v) Construction of DMH9.2 and DMH9.4 pUCll9 was digested with HindIII, and a lkb HindIII
fr~oment, containing a kanamycin resistance gene, purified from 43, was ligated to the HindIII digested pUCll9 to form pMH8 (Figll~e 31). A 2kb SalI fragment (bp 3226-5310), which carries the attP and integrase gene from SalI digested pMH5, was purified ~nd inserted in both orientations relative to the vector backbone ~f ~]~ digested pMH8 to form plasmids pMH9.2 and pMH9.4 (Figures 3~ 33).
M. smegmatis strain mc2155 cells carrying, as a result of e].~troporation~ plasmid pYU812, pMH9.2 or pMH9.4, or strain mc26 cell~ carryin~ ~plasmid pMH5, as a result of electroporation as h~ bove described, were grown to saturation in broth with ~c~n~llmycin. Culture~ were then diluted 1:100 into broth without k~ m~cin and grown to saturation. Two further cycles of d~ ion and growth were done, corresponding to about 20 SUBSrITUTE SHEET

W092/21376 2 1 1 0 ~ 8 2 PCT/US92/04S38 g~ner~tions of bacterial growth. Cultures were plated out to single colonies on non-selective plates, and approximately 100 of thes~ colonies were patch plated onto both non-selective and selective plates. The % of colon~es that were sensitive to kan~ycin, thus corre~ponding to the percentage of cells which lost the pla~mid, is given below in Table I.
Table I
X lo~s pYUB12 (mc2155) 35 pMH5 (mc26) 17 pMH9.2 (mc2155) 3 pMH9.4 (mc2155) 0 (vi) Transformation of BCG with DMH9 . 4 The 1.9 kb Sal I fragment, which includes the M. smeamatis attB ~ite àæ hereinabove described was cloned into pUCll9, and the plasmid generated was named pMH-12. (Figure 34).
Gel purified Sal I l.9kb M. smeomatis fragment ~ontaining ;attB (isolated from pMH-12) was used to probe a Southern transfer of~BAm HI digested mycobacterial DNA's, including BCG substrain PAæteur, hown in Figure 35. This demonstrated that there is one Bam HI fragment of BCG that ~trongly hybridizes to the M.
smegmatis attB probe and three hybridize weakly. The ~trongest hybridizing band is the fastest moving band (approximately 1.9 kb)~-The same probe as above was used to probe a BCG cosmidlibr~ry ~provided by Dr. Bill Jacobs) and positive clones were identified. DNA was prepared from several positive clones and an~ly7.ed by restriction analysis and Southern blotting. The 1.9 kb R~m HI fragment (corresponding to the strongly hybridizing b~nd in the Southern blot was identified, gel purified from the co~mid DNA and cloned into pUCll9. The resulting plasmid was n~me~ pMH-15. (Figure 36).
Plasmid pMH-5 and pMH9.4 were electroporated into BCG
P~ste~r. It. was observed that pMH9.4 transforms BCG with high SUBSTITUTE SHEET
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211 u6~2 _30_ efficiency (approximately 10 transformants/~g DNA), while pMH-5 tr~næforms BCG at low efficiency (1-10 transformants/~g DNA).
DNA wa~ prepared from BCG transformant# and analyzed by Bam HI
reætriction and Southern blot analy~i~, probing with gel purified 1.9kb Bam HI BCG attB fragment from pMH-15. These data are shown in Figure 37 and ~how that integration of both pMH5 and pMH9.4 is ~pecific to the BCG attB site (ie the strongly cross-hybridizing fragment in BCG). This is illustrated by the loss of the l.9kb Bam HI fragment from the transformants and the appearance of two new bands repre~enting attL and attR jun~tion fragments. Figure 37 SllOWS just one of the pMH5/BCG transformants, although all of the four that were analyzed show that one of the band~ (the largest) is smaller than expected (and different in each of the transformants), indicating that the transformation effiency of pMH-5 is low in BCG. In contrast, the four pMH9.4 transformants ~re identical to each other (Figure 37) and give attR and attL
jun~tion fragments of the predicted sizes.
~-~ Plasmid pMH9.4, which includes the mycobacterial phage L5 attP ~ite and the L5 integrase gene, was digested to completion : ` :
with either KpnI ~ PvuII or XbaI ~ PvuII, and a restriction fr~gment of lB62 or lB47 base pairs, respectively, each of which cont~in the attP site and the integrase gene, were purified by ~garo~e gel electrophoresis. Plasmid pMV261~LZ was digested with Xb~ or DraI to genorate either a 7S69 bp or 7574 bp vector fr~ment. The 7569 bp fragment was ligated to the 1862 bp fr~ent derived from pMH9.4 to form pMV460/LZ. The 7574 bp fr~g~ent was ligated to the 1847 bp fragment derived from pMH9.4 to form pMV460 R/LZ. Plasmids pMV460 F/LZ and pMV460R/LZ each i.n~]ll~e a mycobacterial replicon, the L5 attP site, and the L5 int~rase gene. A schematic of the formation of plasmids pMV460 F~l.Z ~nd pMV460R/LZ is shown in Figure 38. To generate ~eri~tives without the mycobacterial plasmid replicon, plasmids pMV~hO~LZ and pMV460/LZ were digested with NotI and re~jr~l~larized by ligation to generate pMV360F/LZ and pMV360R/LZ.

SUBSTlTUTEStlEET

W092/21376 2 1 1 0 S ~ 2 PCT/US92/~538 _ A s~hematic of the construction of pMV360F/LZ and pMV360R/BZ is shown in Figure 39.
Plasmids pMH9.4, pMV261/LZ, pMV460/LZ, pMV460/LZ, pMV460/LZ, ~nd pMV460/LZ were then transformed into M. smeomatis and BCG to te~t their ability to replicate autonomously or integrate into th~ M. smeomatis or the BCG chromosome. Transformation with _ __ _.
pM~q.~, pMV261/LZ, pMV360F/LZ, and pMV360R/LZ yielded kananmycin re~ist~nt transformants of M. smeomatis and BCG. Transformants of ~MV261L2, pMV360F/LZ, and pMV360R/LZ were shown to express E.
coli B-galactosida~e by SDS-polyacrylamide gel electrophoresis And X-gal assay. Plasmids pMV460F/LZ and pMV460R/LZ failed to yiel~ )tanamycin resistant transformants, thus indicating that chr~osomal integration of a plasmid carrying sequences mediating a~t~tlomous replication is lethal to mycobacteria.

7. Construction of PMV307.
Plasmid pMV206 was digested with NotI to remove the myc~b~cterial replicon. The resulting 2209 bp fragment, which incllldes the aph (KanR) gene, the E. coli replicon and the mult.iple cloning site, was ligated and recircularized to form pMV205, the construction of which is schematically depicted in Fig~lre 14.
PCR wlth primers XbaI-Att/Int and NheI-Att/Int was then perormed on a Sal I fragment from pMH9.4, which contains the nttP site and the L5 integrase gene. The resulting cassette was then digested with XbaI and NheI and a 1789 bp fragment was gel purified. pMV205 was then digested with NheI, and the resulting fr~m~nt was ligated to the 1989 bp fragment obtained from pMH9.4 to form pMV307. A schematic of the construction of pMV307 is shown in Figure 40.

8. Construction of PMV261/HIVl-~P 120.
An SmaI-ClaI antigen gene fragment, or cassette, was ~onstr~lcted by PCR, and cloned between the Bam HI and ClaI
restriction sites of pMV261 to form pMV261/HIV1-gp 120.

~` ` SUBSTITUTE SHEET

wo 92t21376 PCI /USg2/04538 2 1 1 0 6 ~ 2 -32- "`' .
Plasmid pMV2~1/HIVl-gp 120 was transformed into BCG, and the prefi~nce of the corresponding antigen in BCG was verified by the appearance of immunoreactive protein bands in Western blot ~nalysis of BCG recombinant ly~ates.

9~. Construction of ~MV361/HIVl-qP 120 The HIVl-gp 120 antigen gene expre~sion cassette, which includes a promoter sequence and an HIVl-gp 120 gene sequence, w~s excised from the pMV261 derivatives with NotI and a second r~striction enzyme site (Pvu II, Eco RI, Sal I, Cla I or Hind II~) and cloned into the integrating plasmid pMV307 between the NotJ site and a second enzyme site (Pvu II, Eco RI, Sal I, Cla I
ol ~jnd III) to form the plasmid pMV361/HIV-Igpl20. The backbone of tl~is plasmid is shown in Figure 41.
~ lasmid pMV361/HIV-lgpl20 was transformed into BCG and shown to ~xpress the corresponding antigens by Western blot analysis (Fiallre 42) with the appropriate antigen-specific human sera.
ExamDle 4 Cytotoxic T lvmDhocvte res~onses to HIV-l-oD 120 HIV-l gp 120 was expressed in BCG afi a six amino acid fusion prot~ir with BCG hsp 6Q protein using vector pMV361~HIV-l-gpl20, sillg the hsp 60 promoter to control expression.
Two groups of mice were inoculated with 1 X 106 CFU's of recom~inant BCG expressing the gp 120 gene from the integrative pl~smid pMV361/HIV-l-gpl20. One group received the BCG via intr~peritonal in~ection (100 ul) whereas the other group received the BCG by deposition of the dose (10 ul) rubbed into a tai] scratch.
CTL activity was measured at various times after imm~ ization. CTL activity was measured as follows:
Two mice from each group were sacrificed at various times after immunation, and the spleens were removed. Single cell ~uspensions were made and the red blood cells were lyzed w~th ammot~ium chloride. The cells were stimulated in vitro for 5 days with P815 cells that were pulsed with peptide P18, a fifteen SUBSTITUTE SHEET

W092/21376 21~ O G ~ 2 PCT/US92/04538 residue ~ynthetic peptide within HIVl-gp 120. A 4-hour chromium ~Cr51) release assay wafi then carried out using untreated P815 ~nd peptide P18 pul~ed P815 cells as targets. Significant P18-æpecific CTL activity wa~ ob~erved in the mice immunized by t.ail scratch 14 weak~ after immunization. At 16 weeks, CTL
~ctivity was observed in both groups of mice. Upon repeat of thi~ experiement, CTL activity was observed at time points a~
early as 8 weeks after immunization.
ExamPle S
Recombinant BCG transformed with pMV361/HIVl-gp 120 were growo to mid-log pha~e in Dubos media and concentrated by centrifugation. The bacteria were then resuspended in 15X
gly~rol and frozen using a rate contolled freezing apparatus.
The ~cteria were stored at -70C until use (referred to as "v~ine"). A second preparation grown in the same way was not fro7.en and is referred to as a "fresh" preparation. Prior to imm~lnization of animals, the bacteria were resuspended in PBS ~
0.05% Tween 80 to the desired concentration and cup sonicated bri~efly to disperse clumped bacteria. Six week old BALB/c mice wer~ inoculated with a ~ingle dose of 5 x 104 cfu fresh bacteria rmined post inoculation) or 1.5 x 105 frozen bacteria (cletormined pre-inoculation) by tail scratch (t. 8. ) injection.
At 8 weeks post-immunization, splenocytes were harvested from nimals and CTL activity was mea~ured (described below).
~pI~oocytes from unimmunized animals were used as controls in the CTI. ~sæays.
CTL activity was determined as follows:
Splenocytes (ACK-treated, 5 X 106/ml~ were stimulated in vitro in 10 ml in upright T25 flasks by co-~ulture for 5 days with mitomycin C-treated P815 cells (5 X 106/ml) that were pulsed with 250 uq/ml of peptide P18 for one hour. A 4 hr. 51Cr release ass~y was subsequently performed in triplicate using P815 targets witll or without pulsing for 1 hour with 250 ug/ml peptide P18~
Vari~ls effector-tarqet ratios were tested using 5000 . SUBSTITUTE SHEET

W092/213~6 PCT/US92/0453*
2110~2 ~34~
targets/well. Specific lysis was calculated as follows: %
specific ly8is = 100 X [ release by effector cells minus ~pontaneous release/maximal release minus spontaneious releasel.
The results are given in Eigure 42.
Aæ shown in Figure 43, both groups of mice ~howed an increased CTL response at 8 weeks after immunization as compared with unimmunized mice.
Exam~le 6 Recombinant BCG transformed with pMV361/HIVl-gp 120. were grown to mid-log phase in Dubos media and concentrated by centrifugation. The bacteria were then resuspended in 15%
glycerol and frozen using a rate contolled freezing apparatus.
Th~ hacteria were stored at -70C until use (referred to as "v~ccine"). A second preparation grown in the same way was not fro7.et~ and is referred to as a "fresh" preparation. Prior to imm~mization of animal~, the bacteria were resuspended in PBS +
O.05% Tween 80 to the desired concentration and cup sonicated riefly to disperse clumped ba~teria. Six week old BALB/c mice were ino¢ulated with a single dose of 5 x 104 cfu fresh bacteria (determined po8t inoculation) or l.5 x lO5 frozen bacteria (d~termined pre-inoculation~ by tail scratch (t. 8. ) injection.
t. ~ weeks post-immunization, splenocytes were harvestad from ~nim~ls and CTL activity was measured (described below).
CTL activity was determined as follows:
Lymph node cells (5 x 106~ml) were stimulated in vitro in lO
ml ~1~ upri~ht T2~ flasks by co-culture for 5 days with mitomycin C-tr~ted P815 cells (5 X lO6/ml) that were pulsed with 250 ug~ml of p~ptide Pl8 for one hour. A 4 hr 5lCr release assay was s1~ks~quently performed in triplicate using P815 (matched ) or EL4 (mismatched) targets with or without pulsing for l hour with 250 ugjml peptide Pl8. Various effector target ratios were tested ~sin~ 5000 targets/well. Specific lysis was calculated as follows: % specific lysis = lO0 x trelease by effector cells :

~' SUBSTITUTE SHEET

wo g2/2l376 ~ 1 ~ 0 6 8 2 PCT/USg2/04~38 min~l~ spontaneous release/maximal relea~e minus spontaneous releasel.
The results of this assay are given in Figure 44.
As shown in Figure 44, a CTL re~ponse to HIV-l gp 120 using lymph node cells was demonstrated following immunization of m~ce with BCG transformed with pMV361/HIV-1 gp 120.
It i 8 to be understood, however, that the scope of the present invention i8 not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.

.~ .

SUBSTITUTE SHEET

Claims (8)

WHAT IS CLAIMED IS:

1. A method of inducing a CTL response in an animal comprising:
administering to an animal mycobacteria transformed with at least one DNA sequence which encodes a protein or peptide or fragment or derivative thereof which includes an epitope which is recognized by cytotoxic T lymphocytes, said mycobacteria being administered in an amount effective to induce a CTL response in an animal.

2. The method of Claim 1 wherein said protein or peptide or fragment or derivative thereof includes an epitope which is recognized by cytotoxic T lymphocytes induced by an HIV protein or fragment or derivative thereof.

3. The method of Claim 2 wherein said protein or peptide or ffagment or derivative thereof is an HIV protein or fragment or derivative thereof.

4. The method of Claim 1 wherein the mycobacteria are of the species M.bovis-BCG.

5. A composition for inducing a CTL response in an animal, comprising:
mycobacteria transformed with at least one DNA sequence which encodes a protein or peptide or fragment or derivative thereof which includes an epitope which is reocognized by cytotoxic T lymphocytes, and an acceptable pharmaceutical carrier, said mycobacteria being present in an amount effective to induce a CTL response in an animal.

6. The composition of Claim 5 wherein said protein or peptide or fragment or derivative thereof includes an epitope whicll is recognized by cytotoxic T lymphocytes induced by an HIV
protein or fragment or derivative thereof.

7. The composition of Claim 6 wherein said protein or peptide or fragment or derivative thereof is an HIV protein or fragment or derivative thereof.

8. The composition of Claim 5 wherein said mycobacteria are of the species M.bovis-BCG.