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EP0613500A1 - Membran-spezifische heterologe Genexpression - Google Patents

Membran-spezifische heterologe Genexpression

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Publication number
EP0613500A1
EP0613500A1 EP92925134A EP92925134A EP0613500A1 EP 0613500 A1 EP0613500 A1 EP 0613500A1 EP 92925134 A EP92925134 A EP 92925134A EP 92925134 A EP92925134 A EP 92925134A EP 0613500 A1 EP0613500 A1 EP 0613500A1
Authority
EP
European Patent Office
Prior art keywords
dna
polypeptide
dna segment
localizing
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92925134A
Other languages
English (en)
French (fr)
Inventor
David W. Niesel
J. Scott Moncrief
Linda H. Phillips
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Texas System
Original Assignee
University of Texas System
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Filing date
Publication date
Application filed by University of Texas System filed Critical University of Texas System
Publication of EP0613500A1 publication Critical patent/EP0613500A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates generally to the exportation 10 of heterologous polypeptides to discrete regions of a host cell in which it is expressed, to nucleic acid sequences encoding exportation polypeptides, to the preparation of membrane embedded epitopes of immunogenic antigens, and to vectors constructed with selected 15 exportation sequences. More particularly, localized expression of polypeptides may be obtained by providing exportation signals encoded by segments of the disclosed nucleic acids that provide for exportation of expressed heterologous polypeptides to the inner 20 membrane/periplasmic space or the outer membrane surface of a host cell.
  • protective vaccines 35 from epitopes of known antigens of eukaryotic, viral or prokaryotic pathogens by taking advantage of the synthetic capacities of transformed host cells. Examples include tumor specific proteins which might be expressed and utilized to stimulate an immune response. Oral vaccines have stimulated research because of the ease of administration and, more importantly, in some instances the unsatisfactory protection a orded from parenteral injection. Vaccination against cholera, for example, gives short-term protection, thus provoking developmental work toward an oral vaccine that would presumably stimulate mucosal intestinal immunity more efficiently (Sanchez et al. , 1990).
  • Salmonella strains are being studied experimentally as particularly attractive candidates for producing oral live vaccines. Attenuated strains have been shown to elicit immune responses in several animal species
  • Protein expression systems have been developed from Salmonella strains.
  • a cloning vector useful for integrating DNA into the aroC gene on Salmonella chromosomes was used to direct expression of heterologous antigens such as tetanus toxin fragment C and Treponema pallidum lipoprotein (Strugnell et al., 1990).
  • heterologous polypeptide gene products orally administered have elicited a serum antibody response, as for example, the cholera toxin B subunit protein expressed from a recombinant Yersinia enterocolitica strain (Sory and Cornells, 1990) .
  • the response was variable and was directed toward polymeric forms of cholera toxin B.
  • cytoplas ic proteins may not produce a high immunogenic response and heterologous proteins from recombinant DNA molecules expressed cytoplasmically often exhibit a diminished antibody reactivity (Sanchez, et al . , 1990).
  • surface expressed epitopes of bacteria are expected generally to elicit the greatest humoral response; however, factors controlling surface expression of heterologous proteins have not been defined and there is no way to assure that any given fusion protein will localize to a host cell membrane surface.
  • Vaccines are the most cost effective medical intervention known to prevent disease. However, effective vaccines are available for relatively few diseases. Successful immunization against infectious organisms often requires a multicomponent host immune response against a variety of antigenic determinants. Orally administered vaccines, especially live attenuated vaccines, induce specific cell-mediated effector responses and elicit secretory IgA (slgA) responses.
  • slgA secretory IgA
  • SlgA is important because of its effectiveness at mucosal surfaces. SlgA production and cell effector responses are mediated through the delivery of antigens to gut- associated lymphoid tissue (GALT) . Stimulation of GALT can lead to effective cell and humoral defense at all mucosal surfaces and provide systemic protection (1,2).
  • heterologous epitopes it is generally believed that the export of heterologous epitopes to the Salmonella cell surface enhances their immunogenicity (11) .
  • Investigators have used recombinant DNA methods to express heterologous epitopes as inserts in Salmonella flagellin (9) and the lamB encoded polypeptide of E. coli (10) .
  • a significant antibody response to the heterologous surface-expressed epitopes was observed.
  • a limitation of these systems is the relatively small number of epitopes which can be inserted into the laraB and flagellin genes. This is important as single (or few) epitopes may not result in the broad-based immune response which characterizes today's most successful vaccines.
  • the present invention addresses one or more of the foregoing or other problems associated with methods of
  • the invention includes nucleic acid segments
  • Such vectors are suitable for expressing and directing heterologous polypeptides exported to selected areas of the host cell.
  • Transformed cells with surface expressed antigens or epitopic regions are expected to be useful as immunogens
  • nucleic acid segments of the present invention encode amino acid sequences associated with particular targeting of fused heterologous polypeptides to
  • nucleic acid segments defined by SEQ ID NO:l encode a polypeptide product which when fused to a heterologous polypeptide will direct that polypeptide to the outer membrane of a bacterial cell.
  • heterologous polypeptide any polypeptide other than those normally associated SEQ ID NO:l. It is of course understood that such localizing capabilities are realized under conditions when the exportation polypeptide is incorporated into a suitable expression
  • a preferred embodiment of the DNA segment is defined by SEQ ID NO:l. This sequence fused to a p oA sequence encodes a 46 Kda polypeptide.
  • the present invention also includes nucleic acid segments encoding amino acid sequences associated with the transport of heterologous polypeptides to the bacterial inner membrane periplasmic space. Particular embodiments of these sequences are included in the nucleic acid sequences defined in SEQ ID NO:2.
  • a pre erred inner membrane periplasmic space directing polypeptide is a 55 Kda polypeptide encoded by the gene sequence illustrated in Figure 3 and defined in SEQ ID NO: .
  • This preferred embodiment includes gene sequences encoding part of the alkaline phosphatase gene, however, other heterologous genes could be used in place of alkaline phosphatase.
  • nucleic acid sequences While particular nucleic acid sequences have been defined it is nevertheless contemplated that nucleic acid sequences will be found to vary. It is expected that analogous sequences with similar functions may be found in other gram-negative bacteria such as E. coli .
  • the invention concerns expression vectors that are constructed to include any of the DNA segments herein disclosed.
  • DNA may be fused directly with a gene of interest and used in an expression system to produce heterologous polypeptides as hybridization probes for, e.g., identifying related sequences, as primers or even as building blocks for the construction of mutant or variant sequences.
  • a particularly useful application of the DNA segements of this invention is to achieve directed expression of heterologous polypeptides.
  • polypeptides will be expressed on the inner membrane periplasmic space, the outer membrane of the host cell, or on the surface of the outer membrane of the host cell.
  • the pZIP plasmids of Figure 2 and Figure 3 have been constructed.
  • fusion polypeptides are exported to the inner membrane/periplasmic space or to the outer membrane of the host cell.
  • 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-negative host cell.
  • pZIP-OUT is a vector which expresses bipartite fusion which includes 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 p oA gene segment lacking signal and expression segments.
  • a variety DNA segments may be inserted into the phoA segment at suitable restriction sites to create a tripartite fusion.
  • pZIP-IN plasmid shown in Figure 3.
  • This plasmid directs the export of polypeptides to the inner membrane/periplasmic space.
  • the construction of the plasmid is bipartite. Part of the alkaline phosphatase gene lacking signal and expression sequences is fused with a DNA sequence that contains an exportation sequence capable of directing its fusion polypeptide to an inner membrane/periplasmic space.
  • plasmids may include, in addition to the export specifying sequences, resistance genes such as ampicillin or tetracycline resistance genes.
  • 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-OUT plasmids.
  • pZIP-IN additionally encodes a kanamycin resistance gene.
  • An advantage of using the phok fusion is that there are various restriction sites within the phoA gene facilitating the fusion of heterologous gene sequences in frame with phoA and the export specifying sequences.
  • Expression vectors may also include a gene encoding a detectable polypeptide.
  • reporter genes encoding detectable polypeptides include ⁇ - lactamase and alkaline phosphatase genes. Reporter genes may be conveniently fused in frame downstream of the disclosed nucleic acid sequences with or without other DNA ragments/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 particularly preferred for transforming bacterial host cells.
  • bacterial host cells Several types may be employed, most preferred being gram-negative cells such as E. coli, Salmonella and the like.
  • Transformed cells may be selected using various techniques including screening by differential hybridization, identification of fused reporter gene products, resistance markers, anti-antigen antibodies, and the like. After identification of an appropriate clone it may be selected and cultivated under conditions appropriate to the circumstances, as for example, conditions favoring expression.
  • Another aspect of the invention is a method of preparing heterologous polypeptides.
  • the method generally involves preparing one or more of the recombinant vectors herein disclosed, transforming a host cell with the recombinant vector, then selecting a vector containing host cell clone and finally isolating from the clone the desired polypeptide which will be a heterologous protein.
  • useful proteins that might be used in preparing the recombinant vector include alkaline phosphatase, cholera toxin B subunit, fragments of these proteins, or any other desired proteins.
  • recombinant heterologous polypeptides will be expressed in different compartments of the cell.
  • heterologous polypeptides expressed in the inner membrane or periplasmic space isolation of the heterologous polypeptide may be affected by cell lysis and other procedures utilized in the isolation of a desired fusion protein.
  • Heterologous fusion proteins exported to the outer membrane of the host cell may be isolated from the outer membrane directly. Typical procedures include separation of inner and outer cell membranes and then isolation of the fusion polypeptide from membranous material.
  • antigenic proteins are expressed on the surface of the host cell.
  • Selected epitopes of eukaryotic viral or prokaryotic pathogens expressed on the surface of a host cell may be used for vaccine development. Tumor specific genes could be expressed and utilized to stimulate an immune response.
  • Whole cells expressing immunogenic epitopes might be used for agglutination-based screening tests.
  • Surface expressed polypeptides of other organisms might be identified by screening recombinant libraries for specific surface expressed polypeptides.
  • cholera toxin B subunit may be expressed on the surface of a Salmonella harboring the pZIP-OUT plasmid vector hereinabove described.
  • cholera toxin B subunits When expressed from Salmonella strain TA2362 harboring plasmid pRSPl ⁇ , cholera toxin B subunits agglutinated in the presence of specific antibody, indicating exposure of epitopic regions on the external membrane surface of formalin-fixed cells.
  • Antigens or epitope(s) are selected and a gene encoding these moieties is inserted into one or more of the recombinant vectors disclosed.
  • Appropriate host cells are transformed and after screening or trans ormants one is selected which expresses the antigen or epitopes for which a vaccine is desired.
  • Vaccines may then be prepared by a variety of methods.
  • Antigens on the surface of appropriate host cells may be safely administered orally. For example, attenuated Salmonella orally administered could stimulate an immune response on gut mucosa.
  • whole cells or cell fragments containing the membrane-bound antigen may be suitably injected into a mammal to generate an immune response. In any event, it is expected that the immunogenicity of an antigen or epitope may be significantly enhanced when expressed on the surface of a bacterial cell.
  • the invention concerns primers capable of priming amplification of selected portions of disclosed DNA segments.
  • Primers hybridize to DNA and serve as initiation sites for synthesis of a portion of the gene.
  • Nucleotide primers are designed to bind at separate sites on opposing duplex strains thereby defining the intervening sequence as the portion to be amplified.
  • Nucleic acid molecules to be employed as primers whether DNA or RNA will generally include at least a 10 nucleotide segment of the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:2.
  • the 10 base pair size is selected as a general lower limit in that sizes smaller than 10 bases hybridization stabilization may be become a problem. However, as the size of the primer decreases too much below 7-8 bases, non-specific hybridization may occur with other genes having complimentary sequences over short stretches.
  • Primers may be utilized for several purposes. For example, primers may be used to amplify selected portions of the disclosed DNA segments. Certain primer combinations may more efficiently generate DNA encoding polypeptides that more effectively target to inner or outer membranes. Additionally, primers prepared from the disclosed DNA may be used to. amplify regions of DNA from other related organisms in order to identify similar targeting sequences. Once amplified products are obtained probes which referred to nucleic acid molecules employed to detect DNA sequences through hybridization procedures may be employed to detect and isolate selected DNA fragments. Like primers, probes may be DNA or RNA and are generally of similar size usually including at least a 10 nucleotide segment or more, often of 220 or 21 base pairs. Probes may be labeled, for example, by radio labeling, to assist in identification of nucleic acid sequences.
  • kits useful for the expression of fusion proteins are also envisioned comprising separate containers, each having suitably aliquoted reagents for performing the foregoing methods.
  • the containers may include one or more vectors, examples being the vectors of claim 19, particular embodiments of which are shown schematically in Figures 4 and 5.
  • Suitable containers might be vials made of plastic or glass, various tubes such as test tubes, metal cylinders, ceramic cups or the like.
  • 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 so as to minimize handling and subsequent volumetric manipulations.
  • Most practitioners will prefer to select suitable endonucleases from common supplies usually on hand; however, such restriction endonucleases could also be optionally included in a kit preparation.
  • Vectors supplied in kit form are preferably supplied in lyophilized form, although such DNA fragments may also be taken up in a suitable solvent such as ethanol, glycols or the like and supplied as suspensions. For most applications, it would be desirable to remove the solvent which for ethanol, for example, is a relatively simple matter of evaporation.
  • FIG. 1 illustrates the cloning of phoA gene fusion from TnpnoA insertion mutants and construction of tribrid gene fusions.
  • Tnp ⁇ oA is a derivative of Tn5 which encodes E. coli alkaline phosphatase, minus the signal sequence and expression signals, inserted into the left IS50L element (21) .
  • Random transposition of TnphoA results in an active insertion only when the phoA gene sequence is fused in frame downstream of the promoter and export 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 begins at the distal joint (DJ) .
  • kanamycin resistance gene e.g., Baj ⁇ HI
  • phoA gene sequence e.g., -Efi-ndlll
  • Plasmids carrying phoA gene fusions can then be used as exposition vectors (B) .
  • the Sspl and Pvull restriction sites in phoA provide blunt ended sites at which in frame insertions (IF) of a gene of interest (GOI) can be inserted.
  • GOI must also be consistent with the phoA frame at the insertion site.
  • the resulting tribrid gene fusions contain the expression and export signals of the target gene fused in frame with the phoA and GOI sequences.
  • FIG. 2B shows the DNA sequence across the
  • IS50L/p oA sequence was observed.
  • a stop codon in this ORF was observed at position -99. Multiple stop codons in all reading frames were present in sequences -150 to - 200.
  • Two putative translation start codons (AUG) were present at positions -84 and -51.
  • the predicted amino acid sequence of the coding region is shown above the nucleotide sequence.
  • the IS50L and the beginning of the phoA derived sequences are underlined.
  • FIG. 2 schematically shows plasmid pZIP-OUT contains a 4.5 Kb -ffindlll chromosomal fragment from invasion-attenuated S. typhimurium TnphoA insertion mutant TAP 43 inserted into pBR322 at the -Hindlll site. It expresses a 46 Kd PHOA fusion protein which localizes to the outer membrane.
  • Figure 3 shows plasmid pZIP-IN which contains a -Ba-mHI chromosomal fragment from S . typhimurium Tnp oA insertion mutant TAG 28, inserted into pBR322 at the SaraHI site. It expresses a 55 kd PhoA fusion protein ⁇ 'hich localizes to the inner membrane.
  • Figure 4 is an immunoblot analysis of Salmonella membrane preparations using mouse anti-alkaline phosphatase. S. typhimurium TA 2362 harboring pBR322 showed no reaction in the total envelope (TE) .
  • TA 2362 harboring pZIP-OUT showed a 46 Kd PHOA fusion in the TE and after separation of the inner and outer membrane by treatment with 0.5% sarkosyl, the majority of the fusion protein was associated with the outer membrane (OM) .
  • TA 2362 harboring pZIP-IN showed a 55 Kd PhoA fusion protein in the TE and after 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 membrane preparations from an equal amount of cells.
  • Figure 5 is an immunoblot analysis of urea extracts (SURF) using anti-alkaline phosphatase as the primary antibody.
  • S. typhimurium TA 2362 harboring pBR322 showed no reacting polypeptides to the alkaline phosphatase antibodies.
  • TA 2362 harboring pZIP-OUT showed a PhoA fusion at 46 Kd.
  • TA 2362 harboring pZIP-IN showed no reacting polypeptides with the same antisera. Lanes were loaded with an equivalent amount of extract prepared from an equivalent number of whole cells.
  • Figure 6 shows the derivation of plasmid pRSP18 from pZIP-OUT in which the final 294 base pairs of ⁇ txB have been inserted in frame (IF) with the phoA gene sequence at the PvuII site.
  • the ctx gene sequence is from pRIT10810 which encodes the entire ctxB gene (22) .
  • Figure 7 shows the derivation of plasmid pIMB13 from pZIP-IN in which the final 294 base pairs of ctxB have been inserted in frame (IF) with the phoA gene sequence at the Sspl site.
  • the ctxB gene sequence is from PRIT10810 which encodes the entire ctxB gene (22) .
  • 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 as the primary antibody.
  • S. typhimurium TA 2362 harboring pRIT10810 which encodes cytoplasmically expressed CTB showed no reaction.
  • TA 2362 harboring pRSPl ⁇ showed a CTB tribrid fusion protein at 32 Kd.
  • TA 2362 harboring pIMB13 showed no reactivity to anti-CTB antibodies.
  • Lanes were loaded with equal amounts of extract from equivalent numbers of whole cells.
  • Figure 10 is a proposed protocol for insertion of a fragment of 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 sequences from pZIP-IN are shown. The ORF is shown in capital letters.
  • the present invention relates to nucleic acid segments encoding particular polypeptides capable of forming fusion proteins that export to particular areas of a host cell. These nucleic acid segments are useful in constructing vectors that allow expression of heterologous proteins in appropriately transformed host cells.
  • Polypeptides may be localized within the inner membrane/periplasmic space or on the outer membrane surface.
  • Antigens or epitopic regions of antigens localized on host cell membranes have particular potential for vaccine development and antibody production.
  • 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 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 Salmonella export specifying sequences.
  • Figure 1. Several cloning sites are possible which allow maintenance of the proper reading frame and produce tribrid fusion polypeptides which contain Salmonella export specifying sequences, the heterologous gene sequences and phoA gene sequences. Recombinants which export the tribrid fusion protein are selected through the loss of phoA activity and appearance of the predicted fusion polypeptide on the surface of the outer membrane.
  • a tribrid fusion has been constructed which encodes virtually the entire cholera toxin B subunit (ctxB) gene.
  • Figure 6 evaluated its subcellular localization in Salmonella.
  • This fusion polypeptide is expressed on the Salmonella surface as evidenced by: 1) agglutination 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 in cell surface preparations.
  • the DNA of the present invention was isolated from Salmonella typhimurium, strain TAP43, an invasion attenuated strain.
  • Invasion attenuated refers to species which have lost one or more virulence factors affecting the efficiency by which Salmonella invades epithelial cells. Isolation of an attenuated strain of Salmonella was considered useful in developing the present invention because such strains may be used to deliver heterologous 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 of a desired antigen is expected to stimulate a response to that species, as well as to the Salmonella or other host antigens.
  • Tnp oA is a transposon derivative of Tn5 in the phoA gene which lacks a promoter, translation initiation site, signal sequence DNA and the first five amino acids of its protein.
  • TnphoA inserts into a foreign gene in the correct orientation and reading frame, gene fusions are generated, coding for hybrid proteins which have alkaline phosphatase activity if transported beyond the inner membrane. Detection of such activity is generally accomplished with an alkaline phosphatase indicator dye, allowing visualization of colored colonies for successful gene fusions that lead to export of heterologous gene products.
  • Part of the present invention contemplates vaccine preparation and use.
  • antigens or epitopes of antigens, will be readily expressed in localized regions of a host cell using the methods disclosed.
  • Expression vectors incorporating the DNA segment encoding exportation polypeptides directing products to a host cell outer membrane surface are expected to be particularly useful. Epitopic regions of antigens, well exposed at a membrane surface, may elicit high immunogenic responses, providing a route to vaccines or antibody production.
  • vaccines which contain peptide sequences as active ingredients are generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference.
  • such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
  • the vaccines are conventionally administered parenterally, by injection, for example, either ⁇ ubcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol t lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • the proteins may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient t r vaccination. Suitable regimes for initial administr ion and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations. 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 physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
  • Various methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C.
  • agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated (Fab)
  • parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
  • physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
  • the vaccine will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations.
  • the vaccinations will normally be administered from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually 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.
  • the 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 Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.
  • 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 following is a general discussion relating to such use and the particular considerations in practicing this aspect of the invention.
  • prokaryotes are preferred for the initial cloning of DNA sequences and constructing the vectors useful in the invention.
  • strains such as E. coli K12 strain 294 (ATCC No. 31446), E. coli B, and E. ' coli X 1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative rather than limiting.
  • prokaryotes may also be preferred for expression.
  • the aforementioned strains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325) , bacilli such as Bacillus subtilus , or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans , and various Pseudomonas species may be used.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g. ,
  • the pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.
  • promoters most commonly used in recombinant DNA construction include the ⁇ -lactamase (penicillinase) and lactose promoter systems (Chang et al. , 1978; Itakura et al., 1977; Goeddel et al. , 1979) and a tryptophan (trp) promoter system (Goeddel et al . , 1979; EPO Appl. Publ. No. 0036776) .
  • eukaryoti ⁇ 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.
  • Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are available.
  • the plasmid YRp7 for example, is commonly used (Stinchcomb et al. , 1919 ; Kingsman et al., 1979; Tschemper et al . , 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 tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977) .
  • Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et al . , 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al . , 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. .
  • the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Any plasmid vector containing a yeast- compatible promoter, origin of replication and termination sequences is suitable.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973).
  • useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293 and MDCK cell lines.
  • Expression vectors for 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 sites, polyadenylation site, and transcriptional terminator sequences.
  • control functions on the expression vectors are often provided by viral material.
  • promoters are derived from polyoma, Adenoyirus 2, and most frequently Simian Virus 40 (SV40) .
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al . , 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the -Efindlll site toward the -Bgll site located in the viral origin of replication.
  • promoter or control sequences normally associated with the desired gene sequence provided such control sequences are compatible with the host cell systems.
  • An origin of replication may be provided either by construction 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 mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. 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.
  • the DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to S. typhimurium gene sequences.
  • nucleic acid probes of an appropriate length are prepared based on a consideration of the sequence, e.g., as shown SEQ ID NO:l and SEQ ID NO:2 or derived from flanking regions of these genes.
  • the ability of such 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 the presence of pathogenic organisms in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other g ⁇ etic constructs.
  • the preferred nucleic acid sequence employed for hybridizations or assays includes sequences that are complementary to at least a 10 to 40, or so, nucleotide stretch of the selected sequence, such as that shown in Figure 1 or Figure 2, SEQ ID N0:1 or SEQ ID NO:2.
  • a size 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 selective.
  • Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • 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 as the PCR technology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant production.
  • nucleotide sequences of the invention are important for their ability to selectively form duplex molecules with complementary stretches of S. typhimurium gene segments.
  • relatively stringent conditions for applications requiring a high degree of selectivity, one will typically desire to employ relatively low salt and/or high temperature conditions, such as provided by 0.02 M-0.15 M NaCl at temperatures of 50°C to 70°C. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
  • the present invention is envisioned as useful in the cloning of nucleic acids encoding certain exportation polypeptides. Identification of other exportation polypeptides in addition to the 46 kDa and 55 kDa proteins should be possible,using methods analogous to those disclosed herein. One method would be to produce a cDNA library using mRNA obtained from mutant S. typhimurium 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 constructed from S . typhimurium mRNA.
  • a method of preparing variants of the S. typhimurium exportation polypeptides is site-directed mutagenesis.
  • This technique is useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, derived from the 46 kDa or 55 kDa protein sequence, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art as exemplified by publications (Adelman et al . , 1983). As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing et al. , 1981). These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single- stranded vector which includes within its sequence a DNA sequence which encodes an export polypeptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Crea et al . (1978) .
  • This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence.
  • appropriate cells such as E. coli cells
  • clones are selected which include recombinant vectors bearing the mutated sequence.
  • 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 to be limiting as there are other ways in which sequence variants of the exportation polypeptide gene may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents to obtain sequence variants (see, e . g. , a method described by Eichenlaub, (1979) for the mutagenesis of plasmid DNA using hydroxylamine) .
  • TnphoA is a derivative of Tn5 which encodes E. coli alkaline phosphatase, minus the signal sequence and expression signals, inserted into the left IS50L element. Random transposition of TnphoA results in an active insertion only when the phoA gene sequence is fused inframe downstream of the promoter and export signals of a target gene A, Figure 1. Plasmids containing phoA gene fusions can then be used as exposition vectors, Figure 1, (B) .
  • the SSP1 and the PvuII restriction sites in phoA are blunt ended sites at which inframe insertions (IF) of a gene of interest (GOI) can be inserted.
  • IF inframe insertions
  • GOI gene of interest
  • Figure 1 is a schematic representation of typical phoA fusions and illustrating cloning of successful fusions.
  • 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 begins at the distal joint (DJ) .
  • Utilizing restriction enzymes which cut either downstream of the kanamycin resistance gene (e . g . , BamHI ) or the phoA gene sequence (e .g. , HindllX) 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 Salmonella sequences, were cloned into the Hind TL or BaroHI site of pBR322, Figures 2 and 3. Plasmids containing phoA gene fusions were then used as exposition vectors. Cells produced fusion polypeptides 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) .
  • nucleic acid segments isolated from a strain of Salmonella similar functions may be obtained from nucleic acid segments from other Salmonella strains and even other microorganisms.
  • the nucleic acid sequences identified and the corresponding encoded polypeptides are useful in developing methods of producing a wide variety of heterologous proteins as well as expression vectors for localizing polypeptides in selected areas of a host cell.
  • This plasmid contains a chimeric gene including a DNA segment from a strain of Salmonella 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.
  • pZIP-IN Figure 3 is a derivative of pBR322 containing a BamHI fragment encoding alkaline phosphatase activity and kanamycin resistance inserted at the BamHI site.
  • the BamHI fragment was cloned from a chromosomal DNA preparation of the TnphoA insertion mutant TAG28, which was constructed by TnphoA mutagenesis (see above) of S. typhimurium TA2361 (phoN mutant derived from LT2) .
  • Chromosomal DNA was prepared from 50 ml of overnight growth of TAG28 in L-broth with vigorous shaking at 37°C.
  • the bacterial culture was precipitated and washed once in phosphate buffered saline (pH 7.0).
  • the washed bacterial pellet was resuspended in 10, ml of ice cold ET buffer [10 mM EDTA, 10 mM Tris-HCl (pH 8.0)]. Lysozyme was added to a concentration of 0.1 mg/ml and incubated for 15 minutes at 37 ⁇ C.
  • sarkosyl-pronase solution (10% sarkosyl, 5 mg/ml pronase in ET buffer) was added and the solution was incubated for 1 hr at 37°C. The solution was then extracted 3 times with TE [(10 mM Tris HCl, 1 mM EDTA (pH 8.0)] saturated phenol followed by 3 extractions with chloroform:isoamyl alcohol (24:1). The aqueous phase was transferred to a 50 ml beaker on ice and one- half volume of 7.5 M ammonium acetate was added. Three volumes of ice cold absolute ethanol was gently layered on top of the solution.
  • the chromosomal DNA was precipitated onto a glass rod by gently stirring the solution to mix the interface.
  • the precipitated DNA was rinsed once in 70% ice cold ethanol and dissolved overnight in 2 ml of TE buffer at 4°C.
  • the concentration of DNA was quantitated by measuring the O.D. at 260 nm.
  • TAG 28 chromosomal DNA 2 ⁇ g was digested with BamHI at 37°C 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 removed and the DNA precipitated by the addition of 1/10 volume 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol followed by centrifugation in a microcentrifuge. 0.2 ⁇ g of pBR322 DNA was digested with BaraHI and prepared for ligation as above. Ligation of the vector DNA (pBR322) and TAG 28 chromosomal DNA was performed by overnight incubation at 4°C in 20 ⁇ l of IX commercial (Promega) ligase buffer and 2 U of T4 DNA ligase.
  • pZIP-IN was isolated from the ligation reaction by transformation of subcloning efficiency DH5 ⁇ competent cells. 5 ⁇ l of the ligation mixture was added to 50 ⁇ l of DH5 ⁇ competent cells and incubated on ice for 30 minutes. Cells were heat shocked for 30 seconds by incubating in a 37°C water bath. Cells were cooled on ice for 2 minutes and 0.950 ml of L-Broth was added to the tube. Cells were incubated for 1 hr at 37°C.
  • Transformants with alkaline phosphatase activity and kanamycin resistance were selected by plating 0.1 ml of the bacterial culture on the L-agar plates containing 50 ⁇ g/ml kanamycin and 40 ⁇ g/ml BCIP (5-bromo-4-chloro-3- indolyl phosphate) , followed by overnight incubation at 37°C. The following day, kanamycin resistant colonies were visible and all were blue, indicating the transformants had alkaline phosphatase activity. This was confirmed by alkaline phosphatase assays, Western blotting with monoclonal antibodies to alkaline phosphatase, and DNA sequencing of the fusion joint.
  • Figure 3 shows a partial restriction map of pZIP-IN.
  • the following example illustrates the construction of pZIP-OUT, Figure 2.
  • the plasmid is constructed from a DNA segment of Salmonella and a PhoA DNA segment lacking signal and expression sequences. When expressed from a suitable host cell, the fusion protein is localized to the outer membrane of the host cell.
  • Genomic DNA was isolated from strain TAP43. A 25 ml culture in LB broth was grown overnight at 37°C with shaking. The cells were harvested by centrifugation, and the pellet washed once in PBS. The washed pellet was resuspended in 10 mis of cold TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). One ml of a 1 mg/ml lysozyme solution was added, and the mixture was incubated in a 37°C water bath for fifteen minutes. After this incubation, 1.2 ml of 10% sarkosyl, 5 mg/ml pronase in TE buffer was added, and incubation continued at 37°C for 1- 2 hours, until cell lysis occurred.
  • TE buffer 10 mM Tris-HCl, pH 8.0, 1 mM EDTA
  • the lysate was then extracted twice with an equal volume of phenol, once with phenol/chloroform, and once with chloroform. To the final extraction, a half-volume of 7.5 M ammonium acetate was added. The solution was mixed gently and placed on ice. Two volumes of ice-cold absolute ethanol were layered on top of the lysate, and the chromosomal DNA was collected at the interface by spooling on a glass rod. The spooled DNA was rinsed once in 70% ethanol, and then allowed to dissolve off of the glass rod into TE buffer overnight at 4°. The buffer, containing the dissolved DNA, was then ethanol-precipitated.
  • the purified chromosome was collected by centrifugation and resuspended in a small volume of TE buffer. 1-5 ⁇ g of the purified DNA was restricted with -ffindlll, and then phenol/chloroform extracted and ethanol precipitated. The sample was collected by centrifugation, the pellet washed once with 70% ethanol, and dried under vacuum.
  • Vector pUC18 was also restricted with Hindi , extracted, and precipitated in the same manner.
  • the J ⁇ i-ndlll fragments of the genomic DNA were then ligated into the -ffindlll site of pUC18 with T4 DNA ligase.
  • 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 presence of an active alkaline phosphatase fusion in the transformant, were selected and analyzed by restriction mapping.
  • Transformant 43-17 contained a 4.5 kp Hindlll insert in the pUCl ⁇ vector. 3.1 kp of this insert consisted of phoK sequences, with the remaining 1.4 kp being derived from Salmonella chromosomal sequences.
  • Salmonella-phoK fusion contained within this Hindlll fragment was designated as the pZIP-OUT cassette. This cassette was subsequently cloned into the Findlll sites of the vectors pBR322 and pAT153. The general structure of pZIP-OUT is shown in Figure 2.
  • the following example illustrates how DNA may be fused to the gene segments of plasmid pZIP-IN, shown in this example with a portion of the cholera subunit B gene.
  • pIMB13 is a derivative of pZIP-IN in which the final 294 base pairs of ctxB have been inserted in frame with the phoA gene sequence at the Sspl site.
  • the inserted fragment containing the ctxB gene sequence is from pRIT10810 which encodes the entire ctxB gene.
  • the Sspl site in the pBR322 portion of pZIP-IN was eliminated as follows. 2 ⁇ g of a plasmid preparation of pZIP-IN was digested with Seal 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 precipitated and ligation was performed in 20 ⁇ l of IX ligase buffer containing 1 U of T4 DNA ligase overnight at 4°C.
  • DH5 ⁇ frozen competent cells were transformed with 5 ⁇ 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 ampicillin. Loss of ampicillin resistance encoded by pZIP-IN indicated that the segment from Seal (3844) to -B ⁇ oRV (185) which contained the Sspl site (4168) had been eliminated. The resulting plasmid p ⁇ S28-l contained a single Sspl site in the phoA sequence which generates an in-frame blunt end cut.
  • pIMB13 was constructed from p ⁇ S2 ⁇ -l as follows.
  • the ctxB sequence encoded by pRITlO ⁇ lO contains an Sspl site which generates an in-frame blunt end cut near the 5' end of the structural gene.
  • pRITlO ⁇ O also contains an Sspl site in the pBR322 portion of the vector. Digestion of pRITlOl ⁇ lO with Sspl generates 2 fragments, one of which contains the 3' final 294 base pairs of ctxB. 2 ⁇ g of p ⁇ S2 ⁇ -l and 2 ⁇ g of pRITlO ⁇ lO were digested with Sspl .
  • White kanamycin resistant colonies were picked for isolation and screened for 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 plasmid was designated pIMB13.
  • the following example is an example of a tripartite fusion prepared from plasmid pZIP-OUT.
  • This plasmid may be used to express a fusion polypeptide from suitable host cells.
  • the DNA inserted in this example is a segment from cholera B toxin subunit.
  • pRSPl ⁇ The construction of the trihybrid fusion, pRSPl ⁇ , was accomplished as follows. Plasmid pRITlO ⁇ lO, containing the cholera toxin B gene, was first restricted with Ec ⁇ R and Pstl. The ends generated by these restrictions were repaired with Klenow, and the vector was ligated back together. This created a .8 kp deletion in pRITlO ⁇ lO, eliminating an undesirable Sspl site in the vector. This deleted pRITlO ⁇ lO was then restricted with Hindlll and Sspl. pZIP-OUT (in vector pUCl ⁇ ) was doubly restricted with Hindlll and PvuII.
  • This example illustrates the procedure for extracting and separating bacterial membranes. After isolation of the membrane fragments, they were analyzed for localization of fusion peptides.
  • 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 activity encoded by pZIP-IN and pZIP-OUT was confirmed by spectrophotometric assay using the chromogenic alkaline phosphatase substrate para- nitrophenol phosphate (PNPP) .
  • PNPP para- nitrophenol phosphate
  • One ml of overnight bacterial cultures was pelleted for 15 seconds in a microcentrifuge. The pellet was washed once in 1 M Tris- HC1 (pH 8.0) and resuspended in 1 ml of 1 M Tris-HCl (pH 8.0). The O.D. 600 of the bacterial suspension was recorded. 50 ⁇ l of chloroform and 50 ⁇ l of 0.1% SDS were added to permeabilize the cells. Samples were vortexed briefly.
  • Figure 5 shows an immunoblot analysis of urea extracts using anti-alkaline phosphatase as the primary antibody. No reaction is shown with plasmid pBR322 or with plasmid pZIP-IN. A reaction is shown with plasmid pZIP-OUT, indicating extraction of the alkaline phosphatase fusion protein.
  • the following example illustrates the expression of a ctxB polypeptide from an attenuated Salmonella strain with localization of the ctxB to the surface of the outer cell membrane.
  • the tribrid fusion in pRSPl ⁇ contains a 1.4 kb Salmonella DNA sequence which includes expression export signals, Figure 6.
  • the phoA sequence of the fusion includes approximately 0.6 kb from the phoA fusion joint (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 9 ⁇ amino acids of ctxB at the C terminus which localizes to the outer membrane.
  • the tribrid fusion in a pIMB13, Figure 7 contains a 1.3 kb Salmonella DNA sequence which includes the expression and export signals of the expressed gene.
  • the phoA sequence of the fusion includes approximately 0.2 kb from the phoA fusion joint 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 9 ⁇ amino acids of ctxB at the C terminus which localizes to the inner membrane.
  • Figure ⁇ is a schematic representation of the fusion products.
  • the following example illustrates the procedures contemplated as useful for creating an immune response in a mammal elicited with virulence attenuated Salmonella strains expressing antigens on the surface of the intact cell.
  • CTB is used as an illustration.
  • mice 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 mice/condition will be challenged with the following: Salmonella alone, or Salmonella with cytoplasmically- encoded CTB (pRITlO ⁇ OlO) , or inner (pIMB-13, Fig. 7) or outer (pRSP-l ⁇ , Fig. 6) membrane-expressed tribrid fusion encoding strains. I.P. challenge (5X10 5 cfu) and oral challenge (5X10 8 cfu) will be evaluated. These challenge doses are expected to give optimal results but may require adjusting as necessary.
  • Boosting will be 10 days post-challenge. Mucosal and serum anti-CTB levels will be determined after l ⁇ 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 mucosal adjuvant activity (17) by comparing antibody titers to Salmonella and Salmonella expressing CTB. Since CTB mediates Ig class switching, we will also determine IgA/IgG ratios between the different challenge protocols by ELISA (17) .
  • the adjuvant activity of membrane expressed CTB will be evaluated using a purified antigen (i.e., ovalbumin) (18) for concurrent challenge with Salmonella or Salmonella expressing CTB strains. Additional experiments to further characterize adjuvant activity will be performed as indicated.
  • a purified antigen i.e., ovalbumin
  • This example illustrates a contemplated method of inserting a fragment of HIV gpl60 gene into plasmid pZIP- OUT of Example 2.
  • a clone containing a 3.1 kb Sail - Xhol fragment encoding the HIV gpl60 gene has been obtained.
  • the coding regions of gpl20 and gp41 are indicated by the arrow in Figure 10.
  • PvuII digestion of this fragment will yield a 1.8 kb fragment which deletes 0.7 kb of gpl20 coding sequence.
  • the 4.5 Kb pZIP-OUT cassette, bounded by Hindlll sites, has been cloned into the ITindlll site of vector pAT153 ( ⁇ PVUII site) . This construction has been designated pZIP-OUT-2.
  • pZIP-OUT-2 will be digested with PvuII and Sail, and the PuvII - Xhol HIV fragment ligated into these sites.
  • the 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, ⁇ gpl20/gp41) .
  • pZIP-IN, pZIP-OUT, and pRSPl ⁇ were sequenced by the Sanger dideoxy protocol for double stranded DNA templates.
  • the cultures were harvested by centrifugation.
  • the cell pellets were resuspended in 100 ⁇ l of 50 mM glucose, 10 mM EDTA, and 25 mM Tris-HCl, pH ⁇ .O, and incubated at room temperature for 5 minutes.
  • the DNA precipitates were collected by centrifugation and each DNA pellet was resuspended in 16 ⁇ l deionized water, 4 ⁇ l 4 M NaCl, and 20 ⁇ l 13% polyethylene glycol 8000. The samples were mixed well and incubated on ice for 20 minutes.
  • the DNA pellets were collected by centrifuging for 15 minutes. The pellets were then washed once with 70% ethanol and dried.
  • the sequencing gel was fixed in 10% methanol, 10% acetic acid, for 1 hour and then dried under vacuum for 1 1/2 hours. 10. The dried gel was then exposed to autoradiograph film at room temperature for -16 hours.

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