CA2123675A1 - Membrane expression of heterologous genes - Google Patents

Abstract

2123675 9310246 PCTABS00022 The invention relates to nucleic acid segments useful in the construction of expression vectors for expression of heterologous polypeptides directed to particular areas of the host cell. Selected constructs direct production of polypeptides to the outer membrane surface of the cell. Other constructs direct expression of heterologous polypeptides to the inner membrane/periplasm of the host cell. Transformed host cells are potentially useful for the production of vaccines or immunogens elicited in response to antigens expressed on the outer membranes of the host cells.

Description

WO g3/10246 PCI`/US92/09~;9 212367 j pE8CRIPTIQN

MB~RU~E E~PRE813IQN_Q~HETEROLOGOltJI~ GENE~

S BAC~GROIJND OF ~!HE I~ JTION

Field of the Invention The invention relates gen~r~lly to the exportation of heterologous polypeptides to di~crete regions of a host cell in which it is expressed, to nucleic acid ~eguences encoding exportation polypeptides, to the preparation of membrane embedded epitopes of immun~genic antigen~, and to vectors c~nstructed with ~elected lS exportation sequence~. More particularly, localized ex~ression of polypeptides may be obtained by providing exportat~on signals encoded by segment~ of the di~closed n~cleic acid~ that provide for exportation of expre~ed heterologous pslypeptides to the inner membrane/peripla~mic ~pa~e or the outer membrane ~urface of a ho~t cell.

~escrimtion of Rela~ed Art Recombinant gene technology has been extensi~ely i~vestigated in the context of expression o~ foreign proteins in host cells whi~h harbor recombinant genes, typically bacterial ho t cell~ 8uch expression is desirable for producing high value protein~, immunogenic polypeptides, and in obtaining hybrid proteins t~at are otherwise difficult to synthesize.

of particular interest is vaccine development. It i~ potentially feasible to prepare protective vaccines ~rom epitopes of kn~wn antigens of eukaryotic, viral or prokaryotic pathogens by taking advantage of the synthetic capacities of transformed host cells. Examples WO 93/10246 PCI'/US92/09659 include tumor specific proteins which might be expressed and utilized to ~timulate an immune response. Oral vaccines have stimulated re~earch because of the ease of administration and, more importantly, in some instances the unsati~actory protection afforded from parenteral in~-ction. Vaccination against cholera, for example, g~ve~ ~hort-ter~ protection, thus provoking developmental work toward an oral vaccine that would presumably stimulate mucosal inte~tinal immunity more efficiently ~Sanchez et al., 1990).

S~l~onella strains are being studied experimentally as particularly attractive candidate~ for producing oral live vaccines. Attenuated strains have been shown to licit im~une respon~es in several an~al spQcie~
(8t~ugnell et ~1., 1990) and apparently can be highly l~unogenic in the ho~t. ~umoral antibody re~ponses includ~ng 108~1 secretory antibody and cellular immune re~pon~e~ have been ob~erved after oral intake (Dougan et d ., 1986). Attenuated ~utant~ have been identified via ~¢reening procedure~ ~uch a~ TnphoA mutagenQs~s, which exclude elimination of mutations in non~ecreted proteins (Niller st ~1., 1989). However, TnphoA method~ only indicate asses~ment of integration of the trangposon into a gene for a secreted or cytoplasmic protein.

Protein expression systems have been developed from Salmonella strains. A cloning vector useful for ~ntegr~ting DNA into the ~roC gene on Salmonella cbromo~omes wa~ used to direct expression of heterologous ~ntigens such as tetanus toxin fragment C and Treponema pall~dum lipoprotein (Strugnell et al., 1990). In some cases, heterologous polypeptide gene products orally ad~ini~tered have elicited a serum antibody response, as 3S ~or exa~ple, ,the cholera toxin B subunit protein expre~sed from a recombinant Yersinia enterocolitica strain (Sory and Cornelis, 1990). Unfortunately, while

2 1 2 3 6 7 ) PCI'/US92/09659 antibodies were detected in sera of challenged mice, the response was variable and wa~ directed toward polymer$c forms of cholera toxin B.

S It i~ recognized that cytopla~mic proteins may not produce a high immunogenic re~ponse and heterologous protein~ from recombinant DNA molecules expressed cytopla~mically o~ten exhibit a diminished antibody reactivity ~8anchez, ~t al., 1990). Thus ~urface expre~ed epitopes of bacteria are expected generally to lioit the greatest humoral re~ponse; however, factors controlling ~urfacQ expression of heterologous proteins have not been defined and there is no way to assure that any given fusion protein will localize to a ho~t cell lS me~brane sur~ace.
.

VaccineR are the most cost ef~ective medical intervention known to prevent disease. However, effec*ive vaccine~ are avail~ble for relatively few di~ea~es. 8uccessful im~unization again~t infectiou~
organism~ oft~n requires a multicomponent ho~t immune recponse against a variety of antigenic determin~nts.
Ora}ly admini~tered vaccines, especially live attenuated ~accines, induce specific cell-~ediated effector re~pon~e~ and elicit ecretory IgA (sIgA) respon~e~.
SIgA i~ ~mportant bec~u~e o~ its e~fecti~ene~s at muco~al surface~. STgA production and cell e~fector responses are mediated through the delivery of antigens to gut-a~sociated lymphoid ti~sue (GALT). Stimulation of GALT
can lead to e~fective cell and humoral defense at all mNco~al surfaces and provide systemic protection (1,2)~

To deliver antigens to GALT, investigators have developed avirulent and virulence-attenuated Salmonella ~tain~. Aromatic dependent (aroA (3)), phoP (4), galE
~5), and cya/crp (6) Salmonella mutants have been reported to interact with GALT in the lamina propria and W093/10~K PCT/US92/~Sg st~mul~te an immune respon~e While it is clearly desirable to use avirulent Salmonella ~trains as carriers for placmids which expre~Q protective antigen~ of other pathogens on their surf~ce, it i~ clear that improvements are needed to develop protective v~ccine~ based on this ~y~te~

~ he u~e of attenuated Salmonella ~train~ to express hetQrologous antigens and sti~ulate GALT is being ~0 ~xten-ively invostigated In some studies, detectable leVel8 of 8pecific mucosal and ~erum antibodies to the h t rologously expressQd antigen have been observed ~7-10) However, in general results with mo~t antigens have ~sen variable ~It i~ gener~lly believed that the export of -~h torologous epitopes to the S~lmon~ cell ~urface nhanco~ their 1D~unogenicity (11) Inve~tig~tors have u~Qd reco~binant DNA mQthods to expres~ heterologous epitopes as insert~ in S~l~onell~ flagellin (9) and the l~mB encoded polypeptide of E col~ (10) In these studie~, a significant antibody response to the h~terologous surface-expressed epitope~ was ob~erYed. A
limitation of these systems is the relatively small number of epitopes which can be inserted into the lamB
and flagellin genes. T~i8 is important as single (or ~ew) epitopes may not result in the broad-baged immune re~ponse which characterizes today's most successful vaccines.
There is clearly a need to develop effective systems to elicit antibody response and in particular to provide methods of exporting heterologous polypeptides to the surface of appropriate~host cells. Antigenic peptides expre~ed on bacterial host cell surfaces may be signiicant in developing vaccines to such important antigens as cholera B subunit toxin and HIV antigens.

, ' , , WO 93/10246 2 1 2 3 6 7 i PCl/US92/09659 ~RY OF q!~E INVENq!ION

The present invention addressQs one or more of the foregoing or other problems asso~iated with methods of controlling surface expression of heterologou~
polypeptides in ~ ho~t cell ~nd provides in particular a method of directing Qxported polypeptide~ to outer cell membrane surfaces or to inner membrane/cytoplasmic regions. ~he invention include~ nucleic acid segments useful for preparing expression vector~. Such vectors are suitable $or expressing and directing heterologous polypeptide~ exported to selected areas of the host cell.
~ransformed cells with surface exprsssed antigene or epitopic regions ~re expected to be useful as immunogens lS producing an effective immune respon~e.

The nucleic ~cid segments of the present invention en¢ode ~ino acid seguQnces associated with particular targeting o~ fused heterologous polypeptide~ to p~rticular areas of a tran~formed host cell. It has been ~ound for example that nucleic ~cid ~egments defined by 8EQ ID N0:1 encode a polypeptide product which when fused to a heterologous polypeptide will direct that polypeptide to the outer membrane of a bacterial cell.
Z5 By ~eterologous polypeptide i~ mea~t any polxpeptide other than tho~e normally a~ociated SEQ ID N0:1. It ic of course understood that such local~zing capabilitie~
are reali~ed under condit~on~ when the exportation polypeptide i~ incorporated into a ~uit~ble expre~sion ~ector and an appropriate cell host is transfonmed with that vector~ A preferred embodiment o the DN~ ~egment i8 defined ~y SEQ ID N0:1. This sequence fused to a phoA
seguence encodes a 46 Kda polypeptide.

Th~ present invention also includes nucleic acid segment~ encoding amino acid sequences associated with the transport of heterologous polypep~ides to the W093/l02~ PCT/USg2/~59 bacterial inner membrane periplasmic space. Particular embodiments of these sequences are included in the nucleic acid sequences defined in SEQ ID NO: 2 . A
preferred inner membrane periplasmic space directing polypeptide is a 55 Xda polypeptide encoded by the gene seguence illustrated in F$gure 3 and defined in SEQ ID
N0:2. This preferred embodiment includes gene sequenceg encoding part of the alkaline pho~phata~e gene, however, other heterologous genes could be u~ed in place of alkaline phosphatase.

While particul~r nucleic acid sequence~ have been defined it is nevertheless contemplated that nucleic acid s~guQncos will b~ ~ound to vary. It i8 expected that ~n~logou~ seguenoe~ with similar functions may be found in other gram-negative bacteria such as E. col~.

In certain particular embodiment~, the invention ¢oncerns exp~ession vectors that ~re constructed to include any of the DNA segments herein disclosed. Such DNA ~ay be fused dire~tly with a gene of interest and used in an expre~sion sygtem to produce heterologous polypeptides ac hybridization probes for, e.g., identifying related ~equences, as primers or even as building blocks ~or the construction of mutant or variant sequences. A particularly useful application of the DNA
segements of this invention is to achieve directed ~xpression of heterologous polypeptides. Depending on the DNA segment selected, polypeptides will be expressed on the inner membrane periplasmic space, the outer membrane of the host cell, or on the surface of thé outer membrane of the host cell.
.. ~
In particular embodiments, the pZIP pla~mids of Figure 2 and Figure 3 have been constructed. Depending on the plasmid elected, fusion polypeptides are exported to the inner membrane/periplasmic space or to the ou~er W093/10246 2 1 2 3 6 7 .i PCT/US92/096sg membrane of the host cell. In a preferred embodiment, pZIP-OUT directs the export of fusion polypeptides to the outer membrane and may also direct a heterologous peptide to the external surface of a gram-neg~tive ho~t cell.
S pZIP-OUT is a vector which expresses bipartite fusion which include~ a DNA segment capable of exporting the fusion product to the external membrane of a gram-negative cell. The other part of the chimeric gene is a phoA gene segment lacking signal and expr~ssion segments.
A variety DNA segments m~y be inserted in~o the phoA
~eg~ent at suitable restriction sites to create a tripartite fusion.

Yet another preferred embodiment is the pZIP-IN
- lS pla~ id ~hown in Figure 3. This pla~id directs the export o~ polypeptides to the inner ~embrane/periplasmic ~pa¢~. The construction of the plasmid is bipartite.
Part of the aIk~line phosphatase gene lacking signal and -expression sequen¢eg is fused wi~th a DNA sequence that ¢ontain~ an exportation sequence capable of dir~cting its fusion polypeptide to an inner membrane/peripl~smic sp~ce. There are several restriction sites in the phoA
gene segment into which foreign DNA or fr~gment~ of DNA
may be inserted.
Other components of either of these pl~smid~ may include, in addit$on to the export speci~ying sequences, resistance genes such as ampicillin or tetracycline resist~ncé genes. In addition an E. coli phoA gene may be fused in frame with expression directing DNA
sequences, such as that used to construct the pZIP-IN and pZIP-OVT plasmids. pZIP-IN additionally encodes a kana~ycin resistance gene. An advantage of using the phoA fu~ion is that there ~re variou~ restriction sites wlthin the phoA gene f~cilita*ing the fusion of heterologous gene sequences in frame with phoA and the export specifying sequences.

' W093/102~ PCT/US92/~659 .~ . .

Expression vectors may also include a gene encoding a detectable polypeptide. Typical examples of reporter genes encoding detectable polypeptides include ~- ~
lactamase and alkaline phosphatase genes. Reporter genes may be conveniently fused in frame downstream of the di~clo~ed nucleic acid sequencec with or without other DNA fragment~/segments. Moreover, restriction sites in the gene sequence of the reporter gene may be used for insertion of a desired DNA fragment(s).
Recombinant vectors such as those described are p~rticularly preferred for tran~forming bacterial host cells. Several types of bacterial host cell~ may be employed, most preferred being gram-negative cells such a~ ~. col~, Salmonella and the like.

Transformed cells may be selected using various technique~ including ~creening by differential hybridization, identification of fused reporter gene produc*~, resistance markers, anti-antigen antibodies, and ~he like. After identification of an appropriate clone it may be celected and cultivated under conditions appropriate to the circumstances, as for example, conditions favoring expression.
Another aspect of the invention i~ a ~ethod of preparing heterologous polypeptides. The method generally inYolves preparing one or more of the recombinant vectors herein di~closed, transforming a host c~ll wlth the recombinant vector, then selecting a vector containing host cell clone and finally isolating from the cloné the desired polypeptide which will be a heterologous protein. Examples of useful proteins that might be used in preparing the recombinant vector include alkaline phosphatase, cholera toxin B su~unit, fragments o~ the~e proteins, or any other desired proteins.

W093/10246 2 1 2 3 6 7 ~ PCT/US92/0965g Depending on the particular recombinant vector celected for transforming a host cell, recombinant heterologous polypeptides will be expre~sed in different compartments of the cell. For tho~e heterologous S pol~peptides expressed in the inner membrane or periplasmic space isolation of the heterologous polypeptide may be affected by cell ly8i~ and other procedures utili~ed in the isolation of a desired fusion protein. Heterologous fu~ion proteins exported to the outer ~embrane of the host cell may be i~olated from the out~r membrane directly. Typical procedures include separation of inner and outer cell membrane~ and then isolation of the fusion polypeptide fxom membranous material.
~5 In a preferred embodiment, antigenic proteins are expressed on the surface o~ the host cell. Selected epitopes of euk~ryotic viral or prokaxyotic pathogens expressed on the surfa~e of a host cell may be used for vaccine development. Tu~or specific genes cou'-d be expressed and utilized to stimulate an immune~response.
Whole cells expressing immunogenic epitopes might be used for agglu~ination-based screening tests. Surface expres~ed polypeptides of other organi~ms might be identi~ied by screen~ng recombinant libraries for gpecific surface expressed polypeptides. In another preferred embodiment, cholera toxin B ~ubunit may be expre~d on the surface of a Salmonell~ harboring the pZIP-OUT pla~mid vector hereinabove described. When expre~sed from Salmonella strain TA2362 har~oring plas~id pRS~18, cholexa toxin B subunits agglutinated in the presence of specific antibody, indicating exposure of epi~opic regions on the extexnal membrane surface of formalin-fixed cells.
Another aspect of the invention involves the prepaxation of vaccines. Antigens or epitope(s) are W093~10~K PCT/US92/ ~ 59 , ~elected and a gene encoding these moieties is inserted into one or more of the recombinant vectors disclosed.
Appropriate hoct cell~ are transformed and after screening for transformants one i~ selected which S ~xpresses the ~ntigen or epitope~ for which a vaccine is desir~d. Vaccines ~ay then be prepared by a variety of ~ethods. Antigens on the surface o~ appropriate host cell~ may be ~afely administered orally. For example, attenuated S~l~onell~ orally administered could stimulate an i~une regponse on gut ~ucosa. Alternatively, whole cell8 or cell ~ragmentg containing the ~embrane-bound antigen ~ay be suitably in~ected into a ~ammal to generate an ir~une response. In any e~ent, it i8 xpsctQd that th~ immunogenicity of an antigen or epitope ~ay bs ~ignificantly snhanced when expre~ed on the - ~ur~ace o~ a bacterial cell.

In both im~unodiagno~tic~ and vaccine preparation, it i~ o~ten po~ible and indeed ~ore practical to prepare ~0 antig ns from s~g~ents of a known lm~n;uMxlunic protsin or - polypeptide. Certa~n epitopic region~ ~ay be ~sed to produce responses similar to those produced by the entire antigeni¢ polypeptide. Often however responses to epitopic regions are not 80 strong a~ re~pon~es to the entire polypeptide. HoweYer, ~urface expreg~ion of the~e epitope~ may generate an enhanced immune response.

In other embodiments, the invention concerns primers capable of priming amplification of selected portions of d~clo ed DNA segments~ Primers hybridize to DNA and serve as initiation sites for synthesis of a portion of the gene. Nuc}eotide primers are designed to bind at separate site~ on opposing duplex strains thereby v~ de~ining the intervening sequence as the portion to be ~pli~ied. Nucleic acid molecules to be employed as pri~ers whether DNA or RNA will generaily include at least a lo nucleotide segment of the nucleic acid wos3/lo~K 2 1 2 3 6 7 t) PCT/US92/~59 sequence of SEQ ID NO: 1 or SEQ ID NO: 2 . The 10 base pair size is selected as a qeneral lower limit in that ~izes smaller than 10 bases hybridization stabilization may be become a problem. However, as the size of the primer decr-ases too much below 7-8 bases, non-specif~c hybrld~zation may oc¢ur with other genes hav~ng complimentary sequences over ~hort stretches.

Primers may be utilized for several purposes. For 1~ -example, primer~ may be used to amplify selected portions o~ the disclosed DNA segments. Certain primer co~binations may more effici ntly generate DNA en¢oding polypeptides that ~ore effectively target to inner or outer membranes. Additionally, primQrs prepared from the lS disclosed DNA aay be used to ampli~y regions o~ DNA from i~ other related organism~ in order to identi~y similar -~- targeting sequences. Once amplified products are obtalned probes which referred to nucleic acid molecules eaployed to detect DNA sequences through hybridization procedures may be employed to detect~and i~olate selected DNA ~rag~ents. Like primers, probe~ may be ~NA or RNA
and are generally of similar size usually including at least a 10 nucleotide ~egment or more, often of 220 or 2 base pairs. Probes may be labeled, for example, by radio labeling, to a~sist in identification of nucleic acid seguen¢e~.

As part of the invention, kits useful for the expre~sion of fusion proteins are al~o envi~ioned compsising separate containers, each having suitably aliquoted reagen~s for performing the foregoing methods.
For example, the containers ~ay include one or more ~ectors~ xa~ples being the vectors of claim 19, particular embodiments of whic~ are ~hown schematically in Figures 4 and 5. Suitable containers might be ~ials made o~ plastic or glass,~various tubes such as test tubes, metal cylinders, ceramic cups or the like.
. ~ ~

W093/10246 PcT/uss2/~sg Containers may be prepared with a wide range of suitable aliquots, depending on applications and on the scale of the preparation. Generally this will be an amount that is conveniently handled 80 as to minimize handling and S subsegu~nt volumetric manipulations. Nost practitioners will pre~er to select suitable endonuclea~es from common ~upplies usually on hand; however, such restriction endonucleaQes could also be optionally included in a kit preparation.
Vectors supplied in kit form are preferably supplied in lyophilized fo~m, although such DNA fragments may also be taken up in a suitable solvent such as ethanol, glycol~ or the like ~nd supplied as suspen~ion~. For ~t ~pplic~tion~, it would be de~ir~ble to remove the ~olv~nt which for ethanol, for example, is a relatively ~imple ~atter of evaporation.

BRIEF DE8CRS N TON OF T~ DRA~ING8 Figure l illustrates the cloning of phoA ~ene fusion from TnphoA insertion mutants and congtruction of tribrid gene fw ions. TnphoA is a derivative of TnS which encodes ~. coli alkaline phosphata~e, minus the 6ignal sequence and expression signal~, inserted into the left ISSOL element ~2l). Random trangposition of TnphoA
result~ in an active insertion only when the phoA gene ~eguence i~ fused in frame downstream of the promoter and ~xport signals of a target gene (A). The point at which the phoA sequence joins the target gene is referred to as the fusion joint (FJ). The remaining portion of the gene beging at the distal joint (DJ). Utilizing restriction enzymes which cut either downstream of the kan~mycin re~tance gene (e.g., BamHI) or the phoA gene sequence - 35 (e.g., ~indIII), allows cloning of phoA gene fusions ~if the target gene is not also restricted ("R")). Plasmids carrying phoA gene fusions can then be used as exposition WO93/10246 212 3 6 7 a PCT/US92/~ss vector~ (B). The SspI and PvuII restriction sites in phoA provide blunt ended sites ~t which in frame in~ertions (IF) of a gene of interest ( GOI ) can be in~erted. The GOI must al~o be con~i~tent with the phoA
S fra~e at the in~ertion ~ite. The re~ulting tribrid gene fusions contain the expression and export signals of the target gene fu~ed in ~rame with the phoA and GOI
sequences.

Figure 2B show~ the DNA sequence across the S~l~onell~: :poA fu~ion ~oint in pZIP-OUT. Dideoxy sequencin- (Segu~na~e 2.0 USB Bioche~cals) was u~ed to deter~lne the 353 base pairs (bp) upstre~m of the S~ln~ phoA ~usion ~oint. A ~lngle open reading lS fr~e (ORF) wb~ch wa~ in ~rame with that of the IS50L/p oA ~equence was observed. A stop codon in this ORF wa~ ob~erved at po~ltion -99. Mult~ple stop codons in all reading ~rames were present in sequences -l50 to -200. Two putatlve translation start codons (AUG) were present at positions -84 and -51.~ A putative Prlbnow box ) was present at posi~ion -120. ~he predic~ed amino acid s0guence of the coding region is ~hown above the nucleotide sequence. The IS50L and the beginning of the ' phoA derived ~equences are underlined.
Figure 2 ~chematically shows pla~mid pZIP-OUT
contains a 4.5 Kb ~ndIII chromosomal fragment from ln~asion-attenuated S. typhimur~um TnphoA insertion mutant TAP 43 inserted into pBR322 at the H~ndIII site.
It expresses a 46 Kd PHOA fusion prote~n which localizes to the uter membrane.

i Figure 3 how~ plasmid pZIP-IN which cont~ins a d I chromosomal fragment from S. typhimur~um TnphoA
~n~ertion ~ut~nt TAG 28, inserted into pBR322 at the BamHI sité. It expresses a 55 k~ PhoA fusion protein which localizes to the inner membrane.
,~:
: ' WO93/10246 PCT/USg2/ ~ 59 Figure 4 is an immunoblot analysis of S~lmonella membrane preparations using mouse anti-alkaline phosphatase. s. typhimurium TA 2362 harboring pBR322 showed no reaction ~n the total envelope (TE) . TA 2 3 62 S harboring pZIP-OUT showed a 46 Kd PHOA fusion in the TE
and after separation of the inner and outer membrane by treatm~nt with 0.5% sarkosyl, the majority oS the fusion protein was a~ociated with the outer membrane (OM). TA
2362 harbosing pZIP-IN showed a 55 Xd PhoA fusion protein in the TE and after separation of the inner and outer nembr~ne by treatment with 0.5% sarko~yl, the ma~ority of the fusion protein W~8 found associatQd with the inner membrane (IN). ~ll lanes were loaded with membrane preparations from an equal amount of cell~.
~5 Figure S i8 an i~munoblot analy~i~ of urea extracts (gURF) using anti-alkaline phosphatase as the primary antibady. S. ty1~ub~1r~um TA 2362 harboring pB~322 ~howed no reacting polypeptides to the alkaline phosphatase antibodie~. TA 2362 harboring pZIP-OUT showed a PhoA
fusion at 46 Kd. TA 2362 harboring pZIP-IN showed no reacting polypeptides with the ~ame antisera. Lanes were loaded with an eguivalent amount of extract prepared from~
an eguivalent number of whole cells.
Fi~ure 6 show~ the derivation o~ plasmid pRSPl8^from pZIP-OUT in which the final 294 base pairs of ctKB have been inserted in frame (IF) with the phoA gene sequence at the PvuII site. The ctxB gene seguence is from pRITl0810 which encodes the entire ctxB gene (22).

Figure 7 ~hows the derivation of plasmid pIMB13 from pZIP-IN in which the fin~l 294 ~ase pairs of ctxB have been inserted in frame (IF) with the phoA gene séquence at the S~pI ~ite. The ctxB gene sequence is from pRITl0810 which encodes the entire ctxB gene (22).

wos3/lo246 PCT/US92/09659 2123~7.~

Figure 8 is a schematic representation of the CtxB
fusion from pRSP18 and pIMB13 that results in exportation of the 32 kDa CtxB protein to the outer and inner membranes, respectively.
Figure 9 is an immunoblot analysis of urea extracts (SURF) using affinity purified anti-CTB a~ the primary antibody. S. typh~murium TA 2362 harboring pRITl0810 which encodes cytoplasmically expressed CTB howed no ~0 reaction. TA 2362 harboring pRSP18 showed a CTB tribrid fusion protein at 32 Kd. TA 2362 harboring p: ~13 showed no re~ctivity to anti-CTB antibodies. ~anes were loaded with equal ~mount~ of extract from eguivalent numbers of whole cells.
Figure 10 is a proposed protocol for insertion of a ~ragment o~ HIV gpl60 gene into pZIP-OUT.

Figure 11 shows the sequence of export specific signal in pZIP-IN. Promoter and regulatory sequences are underlined. IS50L and phoA sèquences from pZIP-IN are shown. The ORF is ghown in capital letters.

PBTAI~D DE8C~IPTION OF ~HB PR~FE~p_EMBODINB~T8 The present invention xelates to nucleic acid segments encoding particular polypeptide~ capable of ~orming fusion proteins that export to pasticular areas o~ a host cell. These nucleic acid segments are useful in constructing vectors that allow expression o~
heterologous proteins in appropriately transformed host cells. Polypeptides may be localized within the inner membrane/periplasmic space or on the outer membrane ~ur~ace. Antigens or epitopic regions of antigens localized on host cell membranes have particular potential for vaccine development and antibody production.

WO93/10246 PCT/USg2/~59 A heterologous gene expression system has been developed which utilizes a virulence-attenuated Salmonella as a carrier for a plasmid expression system ~pZIP-OUT) which can direct the products of large 5 segments of heterologous genes to the outer membrane (Fig. 2). Recombinant DNA techniques are utilized to fuse the reading frame of the gene to be expressed with Salmonell~ export specifying sequence~, Figure 1.
Several cloning sites are possible which allow 10 ~aintenance o~ the proper reading frame and prQduce tribrid fusion polypeptides which contain Salmonella ~xport specifying sequenc~s, the heterologous gene sequences and phoA gene sequences. Recombinants which export the tribrid fusion protein are selected through 15 th~ loss o~ phoA act~vity and appearance of the pr~dicted ;~ ~usion polypeptide on the sur~ace o~ the outer membrane.
:: a tribrid ~usion has be~n con~tru¢ted which encodQs virtually the entire cholera toxin B subunit (¢txB) gene, Figure 6, and evaluated its subcellular localization in 20 S~l nella. This fusion polypeptide is expres d on the Salmonella surface as evidenced by: 1) aggl~tination of -~ tribrid fusion expressing strains by anti-CTB antiserum, 2) localization of the fusion polypeptide in the outer membrane, and 3) the presence of the fusion polypeptide 25 in cell ~urface preparations.

The DNA of the present invention was i~olated from Salmonella typh~murium, ~train TAP43, an invasion attenuated strain. Invasion attenuated refers to species 30 which have lost one or more virulence factor affecting - the efficiency by which Salmonella invades epithelial cells. Isolation of an attenuated strain of Salmonella was considered useful in developing the present invention x because such strains may be used to deliver heterologous 35 antigens to the gut of an animal. Salmonella given orally tends to establish an infection in the intestinal mucosa, leading to an immune response. The presence ~f a , ~ .

W093/l0246 PCT/US92/09659 21231~7 ) desired antigen is expected to stimulate a response to that species, as well as to the Salmonella or other host antigens.

The a~proach to screening for protein export signals was to use alkaline phosphatase fusions based on the TnphoA transposon system reviewed by Manoil et al.
~1990). TnpboA i8 a transposon derivative of Tn5 in the phoA gene which lacks a promoter, tran~lation initiation site, ~gnal ~equence DNA and the first five amino acids of its protein. When the transposon, TnphoA, inserts into a for~ign gen~ in the correct orientation and reading ~rame, g~-~e fusiong are generated, coding for hybrid proteins ~hSch have alkaline phosphatase activity ~ tr~n~ported beyond the inner membrane. Detection of such activity ~s generally accompli~hed with an alkaline pho~phata~e ind~cator dye, allowing ~i~ualization of colored colonie~ for ~uccessful gene fusion~ that lead to export of heterologous gene products.
Part of the present invention contemplate~ ~accine preparation and use. In general, it is con~emplated that antigen~ r or epitopes of antigens, will be readily expre~sed in localized regiong of a host cell u~ing the ~ethod~ disclo~ed. Expres~ion vectorC incorporating the DNA segment encoding exportation polypeptides directing products to a host cell outer me~brane surface ar~
expected to be particularly use~ul. Epitoplc regions of antigens, well ~xposed ~t a ~embrane surface, may elicit high immunogenic responses, pro~iding a route to vaccines or antibody production.

General concepts related to ~ethods of vaccine preparation and use are discussed as applicable to 3S preparations and formulations with antigens, epitopes or sub~ragments of such antigens obtained from various W093/lO~K PcT/uss2/~6s9 ~ourceC; for example, cholera B toxin subunit and the like vaccine Pre~ration and use s Preparation of vaccines which contain peptide seguenoe~ as active inqredients i8 generally well understooa in the art, a~ exemplif~ed by U S Patents 4,608,251; 4,60~,903; 4,S99,231; 4,599,230; 4,596,792;
ana 4,578,770, all incorporated herQin by refQrQnce Typically, such vaccine~ are prepared as in~ectables eith~r a~ liguid solution~ or suspen~ions; solid forms suit~ble for solution in, or ~u~pen~ion in, liguid prior to in~ection ~ay also be prepared The preparation may lS al-o b ~ul~ified The acti~ immunoqenic inqrediQnt is oft-n ~ix d with ~xcipien~s whi¢h are phar~aceutically aoc pt bl- and co~patible witb the activ~ i~gr dient ~uitable~xcipients ar~, for example~, water, saline, doxtro~e, glycerol, ethanol, or tbe like, and co bination~ thereof In addition, if aesired, the vaccine ~ay contain ~inor a~ounts of auxiliar~ sub~tances ~UCh a8 v tting or emul ifying agent4,~pH buffering agen~8, or ad~uvants which enhance the effectiveness of the vaccines.
The vaccines are conventionally administered parenterally, by in~ection, for example, either subcutaneou~ly or intramuscularly. Additional formulations which are suitable for other modes of admlnistr~tion include suppositories and, in some cases, o~al formulations. For suppositories, traditional binaers and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be~or-ed from mixtures containing the attive ingredient in the range of 0.5% to 10%, preferably l-Z%.
~, ~; Oral for~ulations include such normally employed excipients as, for example, pharmaceutical grades of W093/10246 2 123 67 i PCT/USg2/~59 mannitol, lactose, ~tarch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
These compos~tions take the form of solut~ons, suspension~, tablet~, pillC, capsule~, ~ustained release formulations or powder~ and contain 10-95% of active ingredient, preferably 25-70%.

The protQins may be ~ormulated into the vaccine as neutral or salt form~. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups o~ the peptide) and which are formed w~th inorganic acids such as, for example, hydrochloric or pho~phoric acids, or such organic acid~ as a¢etic oxalic, tartaric, ~andelic, and the like. 8~1ts formed with the lS free boxyl group~ ~ay also be derived from inorganic b~e~ uch a~, for example, sodium, potassium, ammonium, c~lciu~, or ~erric hydroxides, and such organi¢ ~ase~ as l~opropyl~ ine, trimethylamine, 2-ethylamino ethanol, hi~tidine, procaine, and the like.
The vaccines are administered in a manne~ compatible with the do~age formulation, and in such amount as will be therapeutically effective and immunogenic. The gy~ntity to be administered depends on the sub~ect to be 2~ treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection de~ired. Precise amount~ of active ingredient required to be administered depend on the ~udgment of the practitioner. However, suitable dosage ranges are of the order of several hundred - micrograms acti~e ingredient per vaccination. Suitable r~imes for initial administration and booster shots are a_~o ~ariable but are typified by an initial admini~tration followed by subsequent inoculations or 3S other administrations.

wos3/lo~K PcT/uss2~ss The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid phy~iologically acceptable S ba~e or in a phy~iologically acceptable dispersion, pasenterally, by in~ection or the like. The do~age of the vaccine will depend on the route of administration and will vary according to the size of the host.

Various methods of achieving ad~uvant effect for the vaccine include use of agents ~uch as aluminum hydroxide or phosphate (alum), commonly used as O.OS to 0.1 percent ~olution in phosphate buffesed saline, admixture with ~ynthetic polymers of sugasC (Carbopol) used as 0.25 p-rcent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging bstw on 70 to 101C for 30 second to 2 minute period~
r pe¢tively. Aggregation by reactivating with pepsin treated (Fab) antibodies to album~n, ~ixture with bacterial cells such as C~ pQrvum or endotoxin~ or lipopolysaccharide components of gr~m-negative bacteria, e~Nl~ion in physiologically acceptable oil vehicles ~uch ~ mannide mono-oleate ~Aracel A) or emulsion with a 20 percent ~olution of a perfluorocarbon (Fluosol-DA) used a~ a block substitute may al~o be employed.

In many instances, it will be desirable to have multiple admini~trations of the vaccine, usually not ~xceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at lea~t about three vaccinations. The vaccinations will normally be admini~tered from two to twelve week ~ntervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-S years, u~ually three years, will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies for the supernatant antigens. ~he assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent No~. 3,791,932; 4,~74,384 and

3,949,064, ~s illustrative of these types of assay~.

The invention also contemplates the use of disclosed nucleic acid segments in the construction of expression vectors or plasmids and use in host cells. The ~ollowing is a general discussion relating to such use and the particular considerations in practicing thi~ aspect of the invention.

Host Cell Cultures and V~ctors In general, of course, prokaryotes are preferred for the initial cloning of DNA sequences and constructing the vectors useful in the invention. For example, in addition to the particular strains mentioned in the more specific disclosure below, one may mention by~way of example, strains such as E. coli K12 strain 294 (ATCC No.
31446), E. coli B, and E. coli X 1776 (ATCC No. 31537).
These examples aré, of course, in~ended to be illustrative rather than limiting.

Other prokaryotPs may also be preferred for expres~ion. The aforementioned strain~, as well ac E.
col~ W3110 (F , lambda-, prototrophic, ATCC No. 273325), bacilli ~uch as Bacillus subtilus, or other enterobacteriaceae such as Salmonella -,yphimurium or Serratia marcesans, and various Pseudomonas species may be used.

In general, plasmid vectors containing replicon and control sequences which are derived from species compat~ble with the host cell are used in connection with W093/l0246 PCT/US92/ ~ 59 ~ ~, these hosts. The vector ordinarily carries a replication site, as well as marking ~equences which are capable of providing phenotypic selection in transformed cells. For ex~mple, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.~., ~olivar ot al., 1977). The pBR322 plasmid contains genes for ampicillin and tetracycline re~istance and thus provides easy means for ident~fying transformed cells.
The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can b~ used by the microorganism for expression.

Those promoters most commonly used in recombinant DNA construction include the B-lactamase (penicillinase) ~nd lactose promoter ~y~tems (Chang et al., 1978; Itakura et ~1., 1977; GoeddQl ot ~1., 1979) and a tryptophan (trp) promoter system ~Goeddel et ~1., 1979; EP0 Appl.
~' Publ. No. 0036776). While these are the most ¢ommonly us~d, other microbial promoters have been dis¢overed and util~zed, and details concerning their nucleotide ~eguence~ have been published, n~bling a sk~lled worker to ligate them functionally into plasmid vectors ~Siebwenlist et ~1., 1980). Certain gene~ from prokaryotes may be expressed efficiently in E. coli from their own promoter ~eguence~, precluding the need for addition of another promoter by artificial mean~. '' In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are available. For expression in - Sacch~ro~yc~s, the plas!id YRp7, for example, is commonly used-(Stinchcomb et al., 1979~; Kingsman et al., 1979;
T~chemper et ~1., 1980). This plasmid already contains the~trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in W093/1O~K 2 1 2 3 6 7 ~ PCT/US92/09659 tryptophan, for exa~ple ATCC No. 44076 or PEP4-~ (Jones, 1977). The presence of the trpl lesion as a oharacteristic of the yeast host cell genome then provides an effective environment for detecting S transformation by growth in the absence of tryptophan.

Suitable promoting ~quence~ in yeast vectors include the promoter~ ~or 3-phosphoglycerate kinase ~Hltzman ot ~1., 1980) or other gly¢olytic enzymes (Hess et ~1., 1968; Holland et ~1., 1978), such as enolase, glyoeraldehyde-3-pho~phate dehydrogenase, hexokinase, pyruvate de¢arboxylase, phosphofructokina~e, glucose-6-phosphate isomerase, 3-phosphoglycer~te mutase, pyruvate kina~, trio~ephosph~te isomera~e, phosphoglucose lS l-oD-ra~e~ and glu¢okina~e. In constsu¢ting suitable ~Ypr ~1on~plasmids, thQ ter~lnation sequences associated wlth th 8~ ~Qnes are also ligated into the expre~sion ¢tor 3' o~ the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.

~,,, Other promoter~, which have the additional advantage o~ transcription controlled by growth conditions are the promoter region for alcohol aehydrogenase 2, i~ocytochrome C, acid phosphatase, degradative enzymes a~sociated with nitrogen metabolism, and the a~orementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes re~pon~ible ~or maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and term$nation ~equences is suitable.
.
In addition to microorganisms, c~ltures of cells der$v~d ~rom multicellular organi~ms may also be used as host~. In principle, any such cell culture is workable, whether ~rom vertebrate or invertebrate culture.
,. , However, interest has been greatest in vertebrate cells, ~-- and propagation of vertebrate ceIls in culture (tissue ,' - .

W093/10246 PCT/US92/~K59 culture) has become a routine procedure in recent years ~Tissue Culture, 1973). Examples of such useful host cell lines are VER0 and ~eLa cells, Chinese hamster ovary (CH0) cell lines, and Wl38, BHK, COS-7 293 and MDCK cell S lines. Expression vectors fos such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA
splice ~ites, polyadenylation site, and transcriptional ter~inator sequences.

For use in ~a~malian-cells, the control functions on the expression v~ctors are often provided by viral raterial. For example, commonly used promoter~ are d riv d ~rom polyoma, Adenovirus 2, and most frequently 8~ n Virus 40 (SV40). The early and late promoters of 8V40 virus ~re particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fier~-et ~l., 1978). Smaller or larger SV40 fragments may al~o be us~d, provided there is included the approxim~tely 2S0 bp ~ . , seguence extending from the HindIII ~ite toward the BglI
~ite located in the viral origin of replication.
Further, it is aiso possible, and often desirable, to utilize promoter or control sequenoes normally associated with the desired gene ~eguence, provided &uch control ~eguences are compatible with the ho~t cell 6y~tems.

An origin of replication may be provided either by con~truction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication - mechan~m. If the vector is integrated into the host cell chromo~ome, the latter is often sufficient.

:
: : , W093/10246 2 1 2 3 6 ~ ~ PCT/US92/09659 Also contemplated within the scope of the present invention is the use of the disclosed DNA as a hybridization probe. While particular examples are provided to illustrate such use, the following provides general background for hybridization applications taking advantage of the disclosed nucleic acid sequences of the invention.

~ucleic Acid HYbridization Embodiments In certain aspects, the DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ~bility to specifically hybridize to S. typh~murium gene seguences. In these aspects, nucleic acid probes of an appropriate length ~re prepared based on a consideration of the sequence, e.g., as shown SEQ ~D N0:1 ~nd SEQ ID
N0:2 or derived from flanking regions of these genes.
The ~bility of cuch nucleic acid probes to specifically hybridize to the S. typhimurium gene sequences lend them particular utility in a variety of embodiments. The probes can be used in a variety of diagnostic assays for detecting ~he presenoe of pathogenic organisms in a gi~en~
sampl~. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primexs, or primers for use in preparing other genetic construct~.

To provide oertain of the advantages in accordance with the invention, the preferred nucleic acid sequence e~ployed for hy~ridizations or assays includes ~equences ~hat are compl~mentary to at least a lo to 40, or so, nucleotide ctretch of the selected ~equence, such as that ~hown in Figure 1 or Figure 2, SEQ ID N0:1 or SEQ ID
N0:2. A ~ize of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and W093/~02~ PCT/US92/09659 selective. Molecules having complementary sequences over stretches greater than lo bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and there~y improve the quality and degree of specific hybrid molecules obtained.
~hus, one will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such a~ the PCR technology of U.S. Patent 4,603,10~, or by introducing selected sequences into recombinant vectors for recombinant production.
~5 Accordingly, the nucleotidæ sequences o~ ~he invention are important for their ability to selectively form duplex molecules with compleme~tary stretches of S.
typh~murium gene segments. Depending o~ the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees o~
selecti~ity of the probe toward the target ~equence. For applications requiring a high degree of selectivity, one ' will typically desire to employ relatively stringent conditions to form the hybrids, for example, one will select relatively low salt and/or high temperature conditions, such as provided by 0.02 M-0.15 M NaCl at temperatures of SOoC to 70C. These conditions are particularly celective, and tolerate little, if any, mismatch between the probe and the template or target strand.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer ~trand hybridized to an underlying template, less stringent hybridization conditions are called for in order to allow formation of the heteroduplex. In these WOs3/10~K 2 1 2 3 6 ~ ~ PCT/US92/~Sg circumstances, one would desire to employ conditions such as 0.15 M-0.9 M salt, at temperatures ranging from 20OC
to s5Oc. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of inoreasinq amounts of formamide, which serves to de~tablllze the hybrid duplex in the same manner as incre~d temperature. Thu~, hybridization conditions can be re~dily manipulated, and thus will generally be a method of choice depending on the d~sired result~.
The present invention is envisioned as useful in the clonlng of nucleic acids encoding cestain exportation polypeptides. Identification of othes expostation polypeptldes in addition to the 46 kDa and 55 kDa lS prot lns chould be possible using methods analogous ~o tho~e di~clo~ed herein. One method would be to produce a cDNA ~ibr~ n using mRNA obtained from mutant S.
typbi urluJ strains. Although the production of cDNA
libraries from bacteria is not commonly done because of the usual absence of poly-A tails on prokaryotic messages, a cDNA library could be constructe~ from S.
typ imur~um mRNA.

A method of preparing variants of the S. typhimurium exportation polypept~des is ~ite-directed mutagenesis.
Thi~ technique is useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptide~, derived from the 46 kDa or 55 kDa protein seguence, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and ~est sequence variant~ ~or example, inc~rporating one or more of the foregoing considerations, by introducing one or more nucleotide ~eguence changes into the DNA. Site-specific mutagenesis '~ 35 allows the production of mutants through the use of ~peci~ic oligonucleotide sequences which encode the DNA
equence of the desired mutation, as well as a sufficient W093~l0246 PCT/US92/~59 number of adjacent nucleotides, to provide ~ primer seguence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about s to lO residues on both ~ides of the junction of the sequence being altered.

In general, the technique of site-specific ~0 mutagQnesis is well known in the art as exemplified by publications (Adelman ~t ~1., 1983). As will be appreciated, the technique typically employs a phage ~ector which exi~t~ in both a single stranded and double stranded ~orm. Typical vectors u~eful in site-directed lS mutag necis include vector~ such as the M13 ph~ge (Mb~cing t ~l., 1981). These phage are readily co~wrcially ~vailable and their use is generally well known to thoSQ skilled in the art.

In general, slte-directed mutagenesig in accordance herewith is performed by first obtaining a single-~tranded vector which includes within its sequence a DNA
~equence whlch encodes an export polypeptide. An oligonucleotide primer bearing the desired mutated seguence is prepared, generally synthetically, for example by the method o~ Crea ct al. (1978). This primer ic then annealed with the single-~tranded vector, and sub~ectsd to DNA polymerizing enæymes such as ~. coli polymerage I Klenow fragment, in order to complete the ~ynthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one s~rand encodes the original non-mutated seguence and the second stxand bears the desired mutation. This heteroduplex vector is then u~ed to transform appropriate cells, such as E. coli r; 35 cellg, and clones are selected which include recombinant vectors bearing the mutated sequence.

~ :

W093/lO~K 2 1 2 3 6 ~ 5 PCT/US92/Og659 The preparation of sequence variants of the selected exportation polypeptide gene using site-directed mutagenesis is provided as a means of producing potentially useful exportation species and is not meant S to be limiting as there are other ways in which sequence variants o~ the exportation polypeptide gene may be obtain~d. For example, recombinant veotors encoding the desired gene may be treated with mutagenic agents to obtain seguence variant~ (see, e.g., a method described by Eichenlaub, (1979) for the mutagene~is of plasmid DNA
using hydroxylamine).

I~olation of Salmonell~ DNA segment~ was accosplished by isolation of DNA fragments containing the phoA g ne phoA fu~ion~. TnphoA i8 a derivative of TnS
which ~ncode~ E. c~lt alkaline phosphatase, minus the ~gn~ qu~nce and exprQ~sion signals, inserted into the l~t I850L element. Random transposition of TnphoA
r~ults in an active insertion only when the phoA gene ~0gu~nce i~ fused inframe downgtream of the promoter and export signals of a target gene A, Figure 1.- Plasmids cont~ining phoA gene fusions can then be used as exposition vectors, Figure 1, ~B). The SSPl and the ~vuII re~triction ~ites in phoA are blunt ended sites at which inframe insertions (IF) of a gene of interest (GOI) can be in~erted. The resulting tribrid gene fusions, shown as C in Figure 1, contain the expression and export ~ignal~ of the target gene fused inframe with the phoA
and GOI sequences.
Figure 1 is a schematic representation of typical phoA fusions and illustrating cloning of successful fu~ions. The point at which the phoA sequence joins the ~` target gene is referred to as the fusion joint (FJ). The re~aining portion of the gene begins at the distal joint (DJ). Utilizing restriction enzymes which cut either downstream of the kanamycin resistance gene (e.g., BamHI) W093/1O~K PCT/US92/09659 or the phoA gene sequence (e.g., HindIII) allows cloning of phoA gene fusions, provided the target gene is not cleaved ("R"). The fusion joint, including all the phoA
gene fusions and upstream Salmon~lla sequences, were S cloned into the ~indIII or BamHI site of pBR322, Figures 2 and 3. Plasmids containing phoA gene fusions were then used as exposition vectors. Cells produced fusion polypeptide~ that had alkaline phosphatase activity, indicated by the formation of blue colonies on agar supplemented with the indicator dye (5-bromo-4-chloro-3-indolylphosphate).

The following examples are intended to illustrate the practice of the present invention and are not intended to be limiting. Although the invention i~
d~m~n~trated with nucleic acid segments isolated from a strain of S~lmonell~, similar functions may be obtained ~rom nucleic acid ~egments from other Salmonella strains and even other microorganisms. The nucleic acid sequences identified and the corresponding encoded polypeptides are useful in developing methodsl of producing a wide variety of heterologous proteins as well as expression vectors for localizing polypeptides in selected areas of a host cell.
BXAMP~B 1 ~ he following illuctrates construction of plasmid pZIP-IN (Fiqure 3). This plasmid contains a chimeric gene including a DNA se~ment from a strain of Salmonel ~ a fused with a segment of the alkaline phosphatase gene lacking signal and expression sequences. When expressed in a suitable host cell, the fusion product is localized to the inner membrane/periplasmic space of the host cell.

W093/102~ 2 1 2 3 ~ 7 i PCT/USg2/09659 Pre~aration of ~-ZIP-IN

pZIP-IN, Figure 3, is a derivative of pBR322 containing a B~mHI fragment encoding alkaline phosphatase activity and kanamycin resistance inserted at the BamHI
s~te. The BamHI fragment was cloned from a chromosomal DNA preparation of the TnphoA ~nsertion mutant TAG28, which was constructed by TnphoA mutagenesis (see above) of S. typh~mur~um TA2361 (phoN mutant derived from LT2).
Chromosomal DNA was prepared from S0 ml of overnight growth of TAG28 in L-broth with vigorous shaking at 37C.
The bacterial culture was precipitated and washed once in pho~phate buffered saline (pH 7.0). The washed b~cterial pellet was re~u~pend~d in 10 ml of ice cold ET buffer ~10 mM EDTA, 10 mM Tri~-~Cl (pH 8.0)]~ Ly~ozyme was added to a concentrat~on o~ 0.1 mg/ml and incubated for 15 minutes at 37C. 1.2 ml of ~arkosyl-pronase ~olution ~10%
sarko~yl, 5 mg/ml pronase in ET buffer) was added and the solution was incubated for 1 hr at 37C. The solution was then extracted 3 times with TE ~(10 mN Tr~s HCl, 1 mM
EDTA ~pH 8.0)~ ~aturated phenol followed by 3 extractions with chloroform:isoamyl alcohol ~4~ he aqueous phase was trans~erred to a 50 ml beaker on ice and one-half volume of 7.5 M ammonium acetate was added. Threevolume~ of ice ~old absolute ethanol wa~ gently layered on top of the ~olution. The chromosomal ~NA was precipitated onto a glass rod by gently stirring the ~olution to mix the ~nterface. The precipitated DNA was rinsed once in 70% ice cold ethanol and dissolved ~vernight in 2 ml of TE buffer at 40C. The concentration of DNA was quantitated by measuring the O.D. at 260 nm.

2 ~g of T~G 28 chromosomal DNA was digested with B~mHI at 37C for 2 hrs. The solution was extracted once with TE saturated phenol, followed by 2 extractions with chloroform:isoamyl alcohol (24:1). The aqueous phase was wos3/lo~K PCT/US92/09659 removed and the DNA precipitated by the addition of 1/lo volume 3 M sodium acetate (pH s.2) and 2 volumes of ethanol followed by centrifugation in a microcentrifuge.
0.2 ~g of pBR322 DNA was digested with BamHI and prepared for ligation as above. ~igation of the vector DNA
(p~R322) and TAG 28 chromosomal DNA w~s performed by overnight incubation at 4C in 20 ~1 of lX commercial ~Promega) liga~e buffer and 2 U of T4 DNA ligase.

pZIP-IN was isolated from the ligation reaction by tr~nsformation of subcloning efficiency DHS~ competent cells. S ~1 of the ligation mixture was added to 50 ~1 o~ DHS~ competent cells and incubated on ice for 30 ~inutes. Cells were heat shocked for 30 ceconds by IS ~n¢ubat~ng in a 37C water bath. Cells were cooled on ice ~or 2 minute~ and O.9S0 ml of L-8roth was added to the tube.- Cells were incubated for 1 hr at 37C.
Tr~nsformant~ with alkaline phosphatase activity and Xan~mycin resistance were selected by plating 0.1 ml of the b~cterial culture on the ~-agar plates containing S0 ~g/ml kanamycin and 40 ~g/ml BCIP (5-bromo-4-~hloro-3-indolyl phosphate), followed by overnight incubation at 37C. The following day, kanamycin resistant colonies were visible and all were blue, indicating the transformants had alkaline phosphatase activity. This was confirmed ~y alkaline phosphatase assays, Western blotting with monoclonal antibodies to alkaline phosphatase, and DNA seguencing of the fusion joint.
Figure 3 shows a partial restriction map of pZIP-IN.

E~MPI.E 2 The following example illustrates the construation - of pZIP-OUT, Figure 2. The plasmid is constructed from a DNA segment of Salmonella and a PhoA DNA segment lacking ~ignal and expression sequences. When expressed from a W0931102~ ~ 1 2 3 ~ 7 ~ PCT/US92/096ss suitable host cell, the fusion protein is localized to the outer membrane of the host cell.

Çonstruction of ~ZIP-OUT

Genomic DNA was isolated from strain TAP43. A 25 ml culture in LB broth was grown overnight at 37C with shaking. The cells were harvested by centrifugation, and the pellet washed once in PBS. The washed pellet was resuspanded in 10 mls of cold TE buffer (10 mM Tris-HCl, pH 8.0, 1 mN EDTA). One ml of a 1 mg/ml lysozyme solution was added, and the mixture was incubated in a 37C w~ter bath for fifteen minutes. After this incub~tion, 1.2 ml of 10% sarkosyl, 5 mg/ml pronase in TE
lS bu~er was added, and incub~tion continued at 37C for 1-2 hours, until cell lysis occurred. The ly~ate was then extractQd twice with an equal volume of phenol, once with phenol/chloroform, and once with chloroform. To the ~in~l extraction, a half-volume of 7.5 M ammonium acetate wa~ added. The solution w~s mixed gently and placed on ice. Two volumes of ice-cold ~bsolute ethano~ weré
layered on top of the lysate, and the chromosomal DNA was collected at the interface by spooling on ~ glass rod.
The ~pooled DNA was rinsed once in 70% ethanol, and then allowed to di~solve off of the glass rod into TE buffer overnight at 4~ The buffer, containing the di~solved DNA, was ~hen ethanol-precipitated. The purified chromo~ome was collected by centrifugation and resuspended in a small volume of TE buffer. 1-5 ~g of the purified DNA was restricted with ~indIII, and then phenol/chloroform extracted and ethanol precipitated.
The ~ample was collected by centrifugation, the pellet washed once with 70% ethanol, and dried under vacuum.

Vector pUC18 was also restricted with ~indI, extracted, and precipitated in the sa~ manner. The ~ndIII fragments of the genomic DNA were then ligated W093/10246 PCT/US92/Og659 into the HindIII site of pUC18 with T4 DNA ligase. After ligation, the DNA was transformed into competent DH5~
cells and plated on L-agar supplemented with ampicillin and BCIP (5-bromo-4-chloro-3-indolyl phosphate), both at 40 ~g/ml. Blue colonies, indicating the presen~e of an active alkaline phosphatase fusion in the transformant, were selected and analyzed by restriction mapping.
Transformant 43-17 contained a 4.5 kp ~indIII insert in the pUC18 vector. 3.1 kp of this insert consisted of phoA sequences, with the remaining 1.4 kp being derived from Salmonella chromosomal sequences.

The identity of this clone as a phoA fusion was confirmed not only by restriction analysis, but also by 80uthern blotting, Figure 4, and seguencing. The S~lmonella-poA fusion contained within this ~indIII
~ragment was designated as the pZIP-OUT cas~ette. This cassette was subsequently cloned into the HindI~I sites of the vectors pBR322 and pAT153. The general structure of pZIP-OUT is shown in Figure 2.

EXl~NPLE 3 The following çxample illu~trates how DNA may ~e fused to the gene segments of plasmid pZIP-IN, shown ln this example with a portion of the cholera su~unit B
gene.

Construction of ~IMB13 pIMB13 is a derivative of pZIPoIN in which the final 294 base pairs of ctxB have been inserted in frame with t~e phoA gene sequence at the SspI site. The inserted fragment containing the ctxB gene sequence is from pRIT10810 which encodes the entire ctxB gene. First, the SspI site in the pBR322 portion of pZIP-IN was eliminated as follows. 2 ~g of a plas~id preparation of pZIP-IN was W093/10246 PCT/US92/~59 212367~

dige~ted with ScaI and EcoRV. Both enzymes cut at a single site within the pBR322 portion of the vector and generate compatible blunt ends. The digested DNA was preoipitated and ligation was performed in 20 ~l of lX
liga~e buffer containing 1 U of T4 DNA ligase overnight at 4-C. DH5~ frozen competent cells were transformed with S ~l of the ligation reaction mixture.
.

Transformants were selected on L-agar plates containing 50 ~g/ml kanamycin. Colonies were then replicated to L-agar plates containing 40 ~g/ml ~picillin. Loss o~ ampicillin resistance encoded by pZIP-IN indicated that the sQgment from ScaI (3844) to ~coRV ~185) which contained the SspI site (4168) had been lS ~ n~t~d. The r~sulting plasmid p~S28-1 contained a ~lngie S~pI site in the phoA seguQnce which generate~ an in-~r~ blunt end cut.

pINB13 was constr~cted from pAS28-1 as follow~. The ctxB c~guence encoded by pRIT10810 contains an SspI site which generates ~n in-fr~me blunt end cut ne~r the 5' end of the ~tructural gene. pRIT1080 also con~ain~ an SspI
site in the pBR322 portion of the vector. ~igestion of pRIT101810 with SspI generates 2 fragments, one of which contains the 3' final 294 ba~e pairs of ctxB. 2 ~g of p~S28-l and 2 ~g o~ pRIT10810 were dige~ted with SspI.
Following phenol/chlorofo D extraction, the samples were combined and precipitated with 2 volumes of ethanol.
Ligation of the sample was performed in 20 ~1 of lX
ligase buffer containing 1 U T4 DNA ligase. DH5~ frozen - competent cells were transformed with 5 ~l of the ligation mixture. Transformants were selected on L-~gar plates containing 50 ~g/ml kanamycin and 40 ~gtml BCIP~
Colonies harboring poS28-1 with inserts at the phoA ssp~
~ite appeared white ~ince insertion interrupted the active phoA gene fusion. White kanamycin resistant ~- colonies were picked for isolation and screened for W O 93/10246 PC~r/US92/09659 expression of a ctxB fusion protein by Western blotting of total envelope fractions with affinity purified anti-ctxB. A DH5~ strain harboring a derivative of pZIP-IN
encoding a ctxB gene fusion was identified and the S plasmid was designated pIMB13.

~XAN~s 4 The following example i8 an example of a tripartite fusion prepared from plasmid pZIP-OUT. This plasmid may be used to express a fusion polypeptide from suitable host cellc. The DNA inserted in this example is a segment from cholera B toxin ~ubunit.

Construction of ~RSP18 The construction of the trihybrid fusion, pRSP18, wa~ accomplished as follows. Plasmid pRIT10810, containing the cholera toxin B gene, was first restricted 20 w~th EcoRI and ~stI. The ends generated by these restrictions were repaired with Klenow, and ~he vector was ligated back together. This created a .8 kp deletion in pRIT10~10, eliminating an undesirable SspI site in the' vector. This deleted pRIT10810 was then re~tricted with ~lndII~ and SspI. pZIP-OUT (in vkctor pUC1~) was doubly restricted w~th ~indII~ and PvuII. A 2.0 kp fragment generated ~rom this double restriction, consisting of 1.4 Xp of Salmonella seguence and .6 kbp of phoA, was i~olated and purified after agarose gel electrophoresis.
This 2.0 kp fragment was then unidirectionally ligated into the XlndI~I/SspI digested pRIT10810. This generated an in-frame fusion of the Salmonella-phoA sequences to the ctxB ~equence (pSP-18). This clone was selected for on the ba~is of weak tetracycline resistance (1 ~g/ml in L-agar). To make further manipulations of the plasmid more e~ficient, a kanamycin gene block (Pharmacia) was WOg3/10246 PCT/US92/096S9 212367.~) cloned into the BamHI site of PSP18, resulting in the plasmid construction pRSP18.

E~AMP~E 5 Ihis example illustrates the procedure for extracting and separating bacterial membranes. After i~olation of the membrane fragments, they were analyzed for localization of fusion peptide~.
~0 p~D~ration of Bacterial Membranes ~Total En~elo~e) and ~e~aration into Inner and Outer Membrane Fractions 100 ml of overnight bacterial cultures grown in L-15 Broth with vigorou~ shaking were pelleted and washed lX
~n pho~phate bu~fered ~aline (pH 7.0). Washea pellets were re~uspended in 3 ml of membrane isolation buffer tlO
rM N~P04, 0.5 mM MgS04 (pH 7.0)]. 8amples were sonicated ~or 20 seconds 3 times with cooling on ice in between.
20 Unbroken cells were removed by centrifugation at 7,000 rpm in a BecXman ultracentrifuge SWS5 rotor for 1 hr.
The supernatants were remo~ed and total envelope pellets were rinsed lX in sterile deionized water. Pellets were' re~u~pended ~n 40 ~1 of sterile deionized water. One-25 ~alf (20 ~1) was saved for We~tern analysis of the total envelope. A 5% ~olution of sarko~yl in ~terile deionized water was added to the remaining 20 ~1 to a final concentration of 0.5%. The samples were incubated for 30 f minuteg at room temperature and centrifuged in a 30 microcetrifuge to pellet the non-soluble fraction representing the outer membrane. The ~upernatant was removed for Western analysis of the inner ~embrane fraction. The outer membrane pellet was rinsed once in sterile deionized water and saved for Western analysis.
~' 35 Figure 4 shows immunoblot analysis of membrane preparations using mouse anti-alkaline phosphatase.

W093~l02~ PCT/US92/~659 The follcwing example describes the analysis of alkaline phosphatase activity. For the purpose~ of the present invention, alkaline phosphatase assays were performed to test for enzyme activity in membrane fractions of host cells in which alkaline fusion proteins were expressed.

Alkaline Phosphatase Assays Alkaline phosphata~e activity encoded by pZIP-IN and pZIP-OUT wa~ confirmed by spectrophotometric assay using the chromogenic alkaline phosphatase substrate para-nitrophenol phosphate (PNPP). One ml of overnight bacter~l cultures was pelleted for 15 seconds in a ~icrocentrifuge. The pellet was washed once in 1 N Tris-HCl ~pH 8.0) and resuspended in 1 ml of 1 M Tris-HCl (pH
8.0). m e O.D. 600 of the bacter~al suspension was recorded. 50 ~l of chloroform and 50 ~l of 0.1% ~DS were added to permeabilize the cells. Samples we~ vortexed briefly. 0.1 ml of a 0.4% solution of PNPP in 1 M Tris-HCl (pH 8.0) was added and samples were incubated at 37C. After significant yellow color was observed, 10 ~l o~ 2.5 M XPO4, 0.5 M EDTA was added and ~amples were - placed on ice to stop the reaction. Cellular debris was removed by centrifugation in a microcentrifuge. O.D. 420 of the samples were recorded. The units of alkaline pho~phatase activity were calculated by the following ~ormula:

Units activity =
1,000 X O.D. 420/time of reaction (minutes) X O.D. 600 , Figure S how~ an immunoblot analysis of urea extracts using anti-alkaline phosphatase as the primary an~ibody.
No reaction is shown with plasmid pBR322 or with plasmid WO93~102~ 2 1 2 3 6 7 ~ PCT/US92/09659 pZIP-IN. A reaction is shown with plasmid pZIP-OUT, indicating extraction of the alkaline phosphatase fusion protein.

E~AMP~E 7 The following outlines the general procedure for extracting proteins from bacterial cells.

Urea Extraction of Bacterial Cells Ten ml of overnight stationary phase bacterial cultures grown in L-Broth with vigorous shaking were cooled on ice for 10 minutes and pelleted at 7,000 rpm in a Bec~man J2-21 (JA-17 rotor). The bacterial pellet was washed 3 times in phosphate buffered saline (pH ~.0).
The wa~hed pellet was re~uspended in 0.1 ml of 6 M urea containing 10 mM ~ris-HCl (pH 7.5) and 5 mM EDTA. The su~pension was incubated or ~0 minutes on ice. Bacteria were pelleted in a micxocentrifuge for 1 minute.
Centrifugation of the supernatants wa~ repeated to remove any traces of debris. Supernatants were frozen and 20 ~1 aliquots were used for SDS-PAGE and Western analysis.

2S EX~PI,B 8 The following example illustrates the expressi~n of a ctxB polypeptide from an attenuated Salmonella ~train with localization of the ctxB to the surface of the outer cell membrane.

Pr~parat~~n of Surface ~xpressed Cholera Toxin Subunit B

The tribrid fusion in pRSP18 contains a 1.4 kb 35 Salmonella DNA seguence which includes expression export signals, Figure 6. The phoA sequence of the fusion includes approximately 0. 6 kb from the phoA fusion joint WO93/102~ PCT/US92/09659 (FJ) to the inframe insertion (IF) of ctxB. The ctxB
sequence includes the final 294 base pairs of ctxB
beginning at the inframe insertion site IF. Expression and export result in a 32 kDa tribrid fusion protein including the final 98 amino acids of ctxB at the C
terminus which localizes to the outer membrane. The tribrid fusion in a pINB13, Figure 7, contains a l.3 kb Salmonella DNA sequence which includes the expression and export signals of the expressed gene. The phoA sequence ~0 of the fusion includes approximately 0.2 kb from the phoA
fusion joint FJ to the inframe insertion IF of ctxB. The ctx8 ~equence includes the final 294 base pairs of ctxB
beginning at the inframe insertion site IF. Expression and export result in a 32 kDa tribrid fusion protein ~nclud~ng the final 98 amino acids of ctxB at the C
terminus which localizes to the inner membrane. Figure 8 is a ~chematic representation of the fusion products.

Whole Salmonella TA2362 cells harboring pRSPl8 were ~hown to express cholera B subunit on the outer surface membrane. Antisera to cholera toxin B subunit were prepared. Agglutination of TA2632 harboring pRSPl8 was obtained. No agglutination was observed with strain TA2362 alone.
An immunoblot ana~ysis of the membrane preparations was ~un using affinity purified rabbit anti-CTB. S.
typhimurium TA 2362 harboring pRSPl8 showed a 32 kDa CTB
tribrid fusion protein in the total envelope (TE). Upon separation of the inner and outer membrane by treatment with 0.5~ sarkosyl, the majority of the fusion protein was observed associated with the outer membrane ~OM). TA
2362 harboring pINBl3 showed a 32 kDa CTB fusion protein in the total envelope (TE). Upon separation of the inner and outer membrane by treatment with 0.5~ sarkosyl, the majority of the fusion protein was found associated with the inner membrane (IM). All lanes were loaded with W093/tO246 2 1 2 3 6 7 ~ PCT/US92/0965g membrane preparations prepared fror n equivalent number of cells.

EX~MPLE 9 s The following example illustrates the procedures contemplated as useful for creating an immune response in a mammal elicited with virulence attenuated Sa~m~nella strains expressing antigens on the surface of the intact cell. In this example, CTB is used as an illustra~ion.

Immunoaenic,Responses from Surface-Expr,e~sed CTB

All immune response experimentation will be conducted using CTB responding C57B/6 mice (15,16). An virulence attenuated S. typhimurium aroA phoN strain will be utilized in all experiments. Groups of 10 micelcondition will be challenged with the ~ollowing:
S~l monel l a alone, or Sal mon~l l a with cytoplasmically-encoded CTB (pRIT~08010), or inner tpINB-~3, Fig. ~) or outer (pRSP-18, Fig. 6) membrane-expressed t~ibrid fusion encoding ctrains. I.P. challenge (5X105 cfu) and oral challenge (5X108 cfu) will be evaluated. These challenge doses are expected to give op~imal results ~ut may require adju~ting as neces~ary. Boosting will be 10 days post-challenge. Mucosal and serum anti-CTB level will be determined after 1 and 2 challenge by ELISA (15,16) and by the ability to neutralize cholera toxin activity on adrenal cells (1). It will also be determined if the membrane-expressed CTB tribrid polypeptide retains its potent m~cosal adjuvant activity (17) by comparing an~ibody titers to Salm~nella and Salmo~ella expressing CTB. Since CTB mediates Ig class switching, we will also determine IgA/IgG ratios between the different challenge protocols by ELISA (17). Alternatively, the adjuvant activity of membrane expressed CTB will be evaluated using a purified antigen (i.e., ovalbumin) (18) for concurrent challenge with Salmonell~ or Salmonella expressing CTB strains. Additional experiments to further characterize adjuvant activity will be performed as indicated.
E~AMPLE lo This example illustrates a contemplated method of inserting a fragment of HIV gp160 gene into plasmid pZIP-OUT of Example 2.

Construction of ~ZIP-OUT Encoding a 60 kDa Fraoment of ~IV oD120 A clone containing a 3.~ kb SalI - XhoI fragment encoding the HIV gp160 gene has been obtained. The coding regions of gp120 and gp41 are indicated by the arrow in Figure 10. PvuII dig~stion of this fragmer.t wil~ yield a 1.8 kb fragment which deletes 0.7 kb of gp120 coding sequence. The 4.5 Xb pZIP-OUT cassette, bounded by ~indIII sites, has been cloned intb the ~ndIII site of vector pAT153 (~PvuII site). This construction has been designated pZIP-0UT-2. pZIP-0UT-2 ' will be digested with PvuII and S~1I, and the E~YII -XhoI HIV fragment ligated into these ~itas. ~he tribrid fusion polypeptide predicted from this construction will yield a 82 kd polypeptide (2000-4000 dal, Salmonella:
20,000 dal, phoA: and 60,000 dal, ~gpl~0/gp41)~

The predicted DNA sequence across the phoA fusion junction into gp120 is shown in Figure 10. The p~A::gp120/gp41 reading frame is indicated by the brackets.~ The amino acid sequence across the fusion joint is shown, WO g3/10246 2 1 2 3 fi 7 ~ PCr/US92/Og659 The following outlines general protocols for sequencing.
s Pre~aration of Tem~lates pZIP-IN, pZIP-OUT, and pRSP18 were seguenced by the Sanger dideoxy protocol for double ~tranded DNA
template~. ~

Purified plasmid preparations for sequencing were prepared as follows: -1. Each ~train was grown overnight in 5 ml of L8 broth (containing the appropriate antibiotic) at 37C with vigorous aeration.

2. The cultures were harvested by centrifugation. The cell pellets were resuspended in 100 ~1 of 50 mM
glucose, 10 mM EDTA, and 25 mM Tris-HCl, p~ 8.0, and incubated at room temperature for 5 minutes.

3. 2D0 ~1 of freshly prepared 0.2N NaOH, 1% D5 were added to each sample. The samples were mixed by inversion, and then incu~at d S minutes on ice.

4. 150 ~1 of 3 M potassium acetate (pH 4.8) were added to each sample. The samples were mixed by inversion and incubated for 5 minutes on ice.

5. The samples were then centri~uged for 5 minutes, and the supernatants transferred to fresh tubes. The ~amples were centrifuged a second time for 5 minutes and the supernatants transferred as before.

WO93~10246 PCT/US92/09659

6. RNase A was added to a concentration of 20 ~g/ml, and the samples were incubated at 37C for 20 minutes.

7. Each sample was phenol/chloroform extracted, chloroform extracted, and then ethanol-precipitated.

8. The DNA precipitates were collected by centrifugation and each DNA pellet wa~ resu pended in 16 ~1 deionized water, 4 ~1 4 M NaCl, and 20 ~1 13% polyethylene glycol 8000. The samples were mixed well and incubated on ice for 2~ minutes.

9. The ~amples were centrifuged 10 minutes and the supernatant~ discarded. The pellets were washed twice in 70% ethanol, dried, and resuspended in 20 ~1 o~ dH20.

Denaturation. Anneal~nq and_Sequencinq of Tem~lates For each DNA template prepared as above:~

1. 2 ~1 of 2 m NaOH~ 2 ~M EDTA were added to the entire~
20 ~1 sample and the sample was incubated for 10 minutes at room temperature.

2. The reactions were neutralized by the addition of 4.5 ~1 of 2 M sodium acetate (pH 5.0) and 5.5 ~1 of distilled H2O. The samples were mixed well, and then precipitated with 100% ethanol.

3~ The DNA pellets were collected by centrifuging for 15 minutes. ~he pellets were then washed once with 70~ ethanol and dried.
4. All of the following reagents, except primers and radioactive label, were supplied in the sequenase W093/102~ PCT/US92/09659 212367~

sequencing kit, United States Biochemical Co. The dried pellets were resuspended in 7 ~l dH20, 2 ~l of 5X Sequenase reaction buffer and 1 ~ 20 ng) of the appropriate primer. For sequencing the Salmonella ~equences in pZIP-IN and pZIP-OUT, immediately upstream from the phoA junction, primer l(AGA ATC ACG CAG AGC G) wa~ used. For extended sequencing in the Sal monel l a sequences of pZIP-OUT, primer 2 tTTC AGG AAT GCA TGC) was utilized. To sequence across the phoA: ctxB junction in pRSP18, primer 3(AGC GCG ACC AGT GAA A) was used. The annealing reactions were incubated for 30 minutes at 37C.

5. To each annealing mixture, 1 ~l of .lM
dithiothreitol, 2 ~l of diluted labelling mix, 1 ~l of tS35~-dATP, and 2 ~l of diluted Sequenase enzyme were added. The reactions were mixed and incubated at room temperature for 5 minutes.
6. 3.5 ~l of each labelling reaction were then transferred to each termination mixture tube, containing dideoxy ATP, dideoxy GTP, dideo~y CTP, and dideoxy TTP. The chain termination reactions were allowed to proceed for 5 minutes at 37C.

7. 4 ~l of stop solution were added to each reaction, and the reactions were heated to 75C for 2-5 minutes.
8. The reactions were loaded onto a 6% acrylamide-urea sequencing gel and electrophoresed at 15 mA for 2-6 hours.
5 9. After electrophoresis, the sequencing gel was fixed in 10% methanol, 10% acetic acid, for 1 hour and then dried under vacuum for 1 1/2 hours.

W093/l0246 PCT/US92/096S9 lO. The dried gel was then exposed to autoradiograph film at room temperature for ~16 hours.

WO93/102~ PCT/US92/09659 212367~

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: BOARD OF REGENTS, THE UNIVERSITY ~F
TEXAS SYSTEM

(ii) INVENTORS: NIESEL, David W.
MONCRIEF, J. Scott PHILLIPS, Linda H.

(iii) TITLE OF INVENTION: MEMBRANE EXPRESSION OF
HETEROLOGOUS GENES

(~v) NUMBER OF SEQUENCES: 2 (v) CORRESPONDENCE ADD~ESS:

(A~ ADDRESSEE: ARNOLD, WHITE & DURKEE
(B3 STREET: P.O. Box 4433 (C~ CITY: Houston (D) STAT~: Texas 77210 (E) COUNTRY: US
(F) ZIP- 77210 (Yi) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible ~C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WordPerfec~ 5.1 (vii) GURR~NT APPLICATION DATA:

~A) APPLICATION NUMBER: Unknown (B) F~LING DATE: Unknown (C) CLASSIFICATION: Unknown ~ ,.

(viii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/792,525 US
(B) FILING DATE: 15 November 1991 (C) CLASSIFICATION: 424 (ix) ATTORNEY/AGENT INFORMATION:

(A) NAME: KTTCHELL, Barbara S.
(B) REGISTRATION NUMBER: 33,928 (C) REFERENCE/DOCKET NUMBER: UTFGlllPCT

(x) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 512-320-7200 (B) TELEFAX: 713-789-2679 ~2~ INFORMATION FOR S~Q ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 213 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNE5S: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GTGTAAAAAC ATG GCG CCT CAT TCT TCT GT~ GTT GGA GTT AAT 93 Met Ala Pro His Ser Ser Val Val Gly Val Asn Met Lys Lys Phe Tyr Ser Cys Leu Pro Val Phe Leu Leu Ile WO93/102~ 2 1 2 3 6 7 ~ PCT/US92/09659 GGC TGT GCT CCT GAC TCT TAT ACA CAA GTA GCG TCC TGG ACG l77 Gly Cys Ala Pro Asp Ser Tyr Thr Gln Val Ala Ser Trp Thr 5 GAA CCT TTC CCG TTT TGC CCT GTT CTG GAA AAC CGG 2l3 Glu Pro Phe Pro Phe Cys Pro Val Leu Glu Asn Arg (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 387 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ ~OPOLOGY: linear (x~) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GCG~GCATAA TAaGCCCTAC ACAAATTGGG AGATATATCA TGAAAGGCTG 50 GCTTTTTCTT GTTATCGCAA TAGTTGGCGA AGTAATCGCA ACATCCGCAT lO0 TAAAATCTAG CGAGGGCTTT ACTAAGCTTG CCCCTTCCGC CGTTGT~ATA l50 ATCGGTTATG GCATCGCATT TTATTTTCTT TCTCTGGTTC TGAA~TCCAT 200 TTACAGCCAT TGCCTGGTTG CTTCATGGGC AAAAGCTTGA TGCGT&GGGC 300 TTTGTAGGTA TGGGGCTCAT AGCTGACTCT TATACACAAG ATGCGC~TGT 350 The references listed below are incorporated herein by reference to the extent that they supplement, explain, S provide a background for or teach methodology, techniques and/or compositions employed herein.

Sanchez, J., Johansson, S., Lowenadler, B., Svennerholm, A.M. and Holmgren, J., Res. Microbiol. 141, 971-979 (1990).

Strugnell, R.~., Maskell, D., ~airweather, N., Pickard, D., Cockayne, A., Penn, C. and dougan, G., Gene 88, 57-63 (lsso) .
~5 Dougan, G., Hormaeche, C.E. and Maskell, D.J., Parasite Immunol. 9, 151-160 ~1986).

Sory, M.-P. and Cornelis, G.R., Res. Microbiol. 41, 921-929 (1990).

Claims (24)

New Claim 1

1. An isolated DNA segment encoding an exportation polypeptide, said DNA
segment being identified as (a) SEQ ID No: 1; or (b) a DNA substantially identical in length to (a) which hybridizes to (a) under stringent conditions; or (c) a DNA substantially identical to (a) which is degenerate as a result of the genetic code to the DNA defined in (a) or (b) and which encodes an exportation polypeptide; or (d) a DNA variant of (a) containing one or more nucleotide changes without functional alteration of the exportation polypeptide encoded by (a).

2. The DNA segment of claim 1 wherein the exportation polypeptide localizes a heterologous protein to the bacterial membrane outer surface.

3. The DNA segment of claim 1 and being defined in Figure 2 (SEQ ID NO:1) and encoding a 46 kDa polypeptide which localizes a heterologous protein to a bacterial cell outer surface.

4. The DNA segment of claim 3 wherein the 46 kDa polypeptide localizes a heterologous peptide to the outer membrane external surface of the bacterial cell.

5. A recombinant vector comprising the DNA segment of any one of claims 1-4.

6. The recombinant vector of claim 5 further comprising a gene encoding a desired polypeptide.

7. The recombinant vector of claim 6, wherein the desired polypeptide comprises a detectable polypeptide.

8. The recombinant vector of claim 7, wherein the gene sequence encoding a detectable polypeptide has at least one restriction site suitable for insertion of a DNA
fragment encoding a desired polypeptide.

79. A peptide which when positioned adjacent to a heterologous protein or peptide localizes such a protein or peptide in the outer membrane of a cell, said localizing peptide comprising an amino acid sequence encoded by the DNA segment defined in SEQ ID NO:1.

10. A method of preparing heterologous polypeptides, comprising transforming a cell with the recombinant vector of claim 5 to provide one or more vector-containing recombinant host cells; and culturing the transformed cell to obtain the heterologous polypeptide.

11. A recombinant heterologous polypeptide prepared by the method of claim 10.

12. The method of claim 10 wherein the heterologous polypeptide comprises an antigenic protein or an epitope of said antigenic protein.

13. The method of claim 12 wherein the antigenic protein or epitope of said protein is cholera toxin subunit B.

14. A Salmonella typhimurium transformant prepared by the method of claim 10.

15. A method for preparing a vaccine, comprising the steps:
selecting an antigen or epitopes of said antigen to which an antibody is desired;
inserting a gene encoding the antigen or epitopes of said antigen into the recombinant vector of claim 5;
transforming a host cell with said recombinant vector;
screening for transformants;
selecting a transformant which expresses the antigen or epitopes of said antigen; and preparing a vaccine from the cells expressing the antigen or epitopes of said antigen.

16. The method of claim 15 further comprising isolating outer cell membrane fractions from said selected transformant for preparing the vaccine.

17. The method of claim 15 wherein the host cell is Salmonella typhimurium or Escherichia coli.

18. The method of claim 15 wherein the host cell is a virulence attenuated strain of Salmonella.

19. The method of claim 15 wherein the antigen or epitopes of the antigen are bacterial.

20. The method of claim 15 wherein the antigen comprises cholera toxin B subunit.

21. A set of primers capable of priming amplification of selected portions of the DNA of claim 1.

22. A kit comprising at least one cloning vector in accordance with claim 5, the vector being suitably aliquoted into a container.

23. The kit of claim 22 comprising the cloning vector of claim 5 encoding an exportation polypeptide capable of localizing to outer membrane locations of the host cell.

24. The kit of claim 22 wherein the cloning vector comprises pZIP-OUT which is identified in Fig. 2A.