WO2000044405A1

WO2000044405A1 - Dmsa promoters and derivatives thereof used for controlled expression of foreign genes

Info

Publication number: WO2000044405A1
Application number: PCT/US2000/002153
Authority: WO
Inventors: Nadav Orr
Original assignee: University Of Maryland, Baltimore
Priority date: 1999-01-27
Filing date: 2000-01-27
Publication date: 2000-08-03
Also published as: AU2634100A

Abstract

A novel bacterial vector capable of expressing foreign proteins in vivo is disclosed herein. The expression is specifically controlled by an anaerobically inducible FNR promoter, dmsA or its derivatives, operatively linked to a DNA sequence encoding the protein of interest. The dmsA promoter allows for in vivo expression of foreign proteins, such as vaccine antigens, in live attenuated vaccine strains. Two derivatives of dmsA were engineered to enhance the expression under anaerobic growth conditions by otpimizing the FNR binding sequence. The protein expression driven by these promoters was tightly controlled by anaerobiosis and to a lesser extent, inhibited by nitrate. The use of dmsA promoter derivatives for expression of foreign proteins in live attenuated vaccines offers a modular promoter system for expression of proteins at different levels. This allows tailoring an optimal expression system by matching the level of expression of a specific foreign gene while imposing minimal metabolic stress on the carrier strain.

Description

TITLE OF THE INVENTION: dmsA Promoters and Derivatives Thereof Used For Controlled Expression of Foreign Genes

CROSS REFERENCE TO RELATED APPLICATION: This application is related to U.S. Provisional Application 60/1 17,470 filed on January 27,

1999.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT: The invention described herein was supported by funding from the National Institutes of Health (Grant ROl -A 129471-08). The U.S. government has certain rights in the invention. It was also supported by the University of Maryland, Baltimore. BACKGROUND OF THE INVENTION: FIELD OF THE INVENTION:

The field of this invention generally relates to the controlled expression of desired genes in bacteria. More specifically, this invention relates to the controlled expression in bacteria of desired genes using an anaerobically inducible prokaryotic promoter, dmsA, and derivatives thereof.

This invention also relates to using the dmsA promoter and derivatives thereof to control expression of foreign genes in live bacterial strains. This field of the invention more specifically relates to the targeted delivery and expression of therapeutic proteins to animal host cells using the anaerobically induced dmsA promoters and derivatives. The dmsA promoter and derivatives allow for the controlled manipulation of protein expression in a variety of environments, including anaerobic and nitrate-rich conditions. In the most preferred embodiment, the therapeutic protein is a vaccine antigen and the invasive bacteria is an attenuated vaccine strain carrying a plasmid construct therein.

DESCRIPTION OF RELATED ART: Live vector vaccines, also called "carrier vaccines" or "live antigen delivery systems", comprise an exciting and versatile area of vaccinology (Levine et al, Microecol. Ther., 19:23-32 (1990). In this approach, a live viral or bacterial vaccine is modified so that it expresses protective foreign antigens of another microorganism, and delivers those antigens to the immune system, thereby stimulating a protective immune response. Live bacterial vectors that are being promulgated include, among others, attenuated Salmonella, Shigella, Yersinia enterocolitica, V. cholerae 01 and E. coli. Each has certain advantages and some disadvantages.

Attenuated S. typhi is particularly attractive as a live vector vaccine for humans because it is administered orally, colonizes the gut-associated lymphoid tissue as well as the reticuloendothelial system, elicits a broad immune response (that includes serum antibodies, mucosal SIgA, diverse cell-mediated immune responses including classical CTL, and a form of antibody-dependent cellular cytotoxicity). Moreover, Salmonella are readily manipulated genetically and many foreign antigens have already been expressed in these bacteria. In theory, one-dose oral vaccines against a variety of infectious diseases can be developed by stably introducing and expressing foreign genes encoding protective antigens in a well-tolerated yet highly immunogenic S. typhi live vector strain (Levine et al, Microecol The r 19:23-32, (1990). 22). The possibility of using live attenuated vaccine strains as vectors for introducing foreign antigens is an attractive approach for achieving immunity against several pathogens in a single administration. However, depending on the specific foreign gene, the expression may impose metabolic stress on the already attenuated carrier strain thereby inhibit its growth in vitro as well as limit its immunologic effect in vivo. The optimized system is therefore, the one that will have minimal expression of the foreign antigen while the carrier strain is grown in vitro prior to inoculation and before it reaches its target tissue. Upon introduction into the host, the host environment should trigger the expression in a controlled manner thus enabling the presentation of the foreign antigen together with the native carrier antigens.

An attractive approach for control is to use anaerobic induced promoters, members of the FNR dependent promoters. The advantage of using anaerobic-induced promoters is significant in vaccine strains like attenuated Salmonella typhimirum (S. typhimurium) and Salmonella typhi (S. typhi) that are intended to activate the immune response in anaerobic environment. The FNR modulon represents a family of operons whose expression is regulated by the FNR protein in response to anaerobiosis (10, 15). Over thirty transcription units including over then seventy genes were recognized as members of the FNR-responsive family, most of them are activated by FNR. All of the genes in this family encode proteins that are involved in cellular adaptation to growth under anaerobic environment.

The FNR protein is isolated from cells as a monomeric form of about 28 kDa and the oxygen sensing module comprise of an iron bound to a cluster of four cysteine residues, three of them are located in the N-terminal of the protein. When the protein is exposed to reducing environment it is activated and binds to its target sequence. The typical positively FNR-regulated promoters includes a weak -35 element and a binding sequence containing two FNR binding domains centered at about — 41 -5- A 22 bp FNR-site consensus sequence was deduced from sequence comparisons of the different FNR responsive modulons. In many cases the FNR responsive operons include another regulatory site that response to nitrate (6). The nitrate responsive protein, NarL in its phosphorylated form is thought to act directly as a transcriptional activator or repressor, depending on the location of the NarL binding sequences.

Several systems that use anaerobically induced promoter have been previously described. Most of those systems are based on the nitrate reductase promoter nirB, a member of the FNR promoters family (12). It was shown that m>15, a derivative of nirB, was induced upon invasion into eukaryotic cells and drive the expression of antigen within macrophages (7). By using m>15 it was possible to express foreign antigens from plasmids in Escherichia coli (E coli) (17), as well as in attenuated S. typhimurium and S. typhi vaccine strains (1, 2, 4, 8, 9, 14). Furthermore, when those strains were given to mice orally (S. typhimurium), or intranasally (S. typhi), significant immune responses were induced and protective levels of antibodies were achieved (2, 4, 8). Certain vector vaccines of the prior art suffer from a problem known as "leakiness", wherein the expression of foreign protein is activated on at inappropriate time or in an undesired environment. This problem can be particularly serious when the foreign protein is toxic to certain host cells. This uncontrolled expression of the protein, even if not toxic, can affect the immune response by elevating the background expression level beyond certain desirable limits. A related problem is overexpression, wherein the gene is turned on in the correct environment but at a level too high for the host system to bear. The control of the activation or induction of protein expression is the domain of the particular promoter operatively linked to the DNA sequence encoding the protein. There is a need in the vaccine art for a promoter capable of modulating the timing, level or environment of protein expression to facilitate the desired response.

The invention herein is directed to an anaerobically inducible prokaryotic promoter, dmsA , a member of the FNR dependent family. The dmsA promoter drives the expression of dmsABC operon encoding dimethylsulfoxide reductase (3). The dmsA operon includes FNR binding sequence and three NarL binding sequences downstream to the FNR site. It has been shown that the dmsA is activated in vitro by anaerobiosis and repressed by nitrate (5, 18. 19). The use of dmsA promoter in vivo offers a novel system capable of a controlled activation in vivo for the expression of recombinant genes in live attenuated vaccine strains. SUMMARY OF THE INVENTION

It is the object of the invention to provide an expression vector that utilizes the anaerobically inducible prokaryotic promoter, dmsA, or derivatives thereof, to allow for the controlled manipulation of the expression of the foreign genes contained with a carrier expression vector. It is a further object of the invention to provide novel anaerobically inducible prokaryotic promoters that can be used in combination with bacterial expression vectors allowing for the controlled expression of foreign genes in a variety of in vivo and in vitro environments.

In a preferred embodiment, the expression vector is a prokaryote, more preferably a bacteria. In the most preferred embodiment, the bacterial vector is a live attenuated vaccine strain. In a preferred embodiment, the foreign gene encodes a therapeutic protein. In a most preferred embodiment, the therapeutic protein is a vaccine antigen. It is a further object of the invention to provide a method of controlling the expression of foreign genes in a variety of environments, including anaerobic and nitrate-rich conditions.

It is a further object of the invention to provide an expression vector with a sufficiently high induction ratio to facilitate controlled foreign gene expression in vivo. It is a further object of the invention to provide an expression vector with nitrate response elements, thereby allowing for controlled of expression of the foreign gene in vitro or in vivo.

It is a further object of the invention to provide a prokaryotic promoter with both FNR and NAR binding sequences to allow for the controlled manipulation of expression and control of induction under anaerobic conditions and in the presence of nitrates. It is a further object of the invention to provide an expression vector with a dmsA promoter or derivatives thereof to limit background expression and/or prevent overexpression of the foreign gene contained in the vector.

It is a further object of the invention to address the problem of "leakiness" associated with bacterial expression systems of the prior art. It is an object of this invention to create the DNA consensus sequence for a promoter that is induced in anaerobic conditions.

It is another object of this invention to generate a mutated anaerobically induced promoter having increased promoter activity over the wild-type promoter lacking the mutations.

It is an object of this invention to create a plasmid containing an anaerobically induceable promoter for controlled expression of nucleic acid encoding for a desired product. It is a further object of this invention that the plasmid contains a mutated promoter with enhanced activity over the non-mutated promoter.

It is still another object of this invention to have bacteria containing an expression vector that is induced in anaerobic conditions. BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 : Depicts the published nucleotide sequence of the entire dmsA promoter (SEQ ID NO: 1). The dmsA promoter is 900 bp down stream of Sma I site (3). The FNR binding sequence is double-underlined (bp 594 to bp 615 -- SEQ ID NO: 2). The three NAR-L binding sequences are underlined (bp 616 to bp 622; bp 636 to bp 642: bp657 to bp 663). The arrow mark the transcription initiation point (bp 593). The ATG of DMSA is in bold (bp 869 to bp 871). Figures 2A-C: Depicts the dmsA promoters used in the studies herein (EcoR I, Bgl II fragments). Specifically, Figure 2A depicts the nucleotide sequence of the dmsA promoter (SEQ ID NO: 3). Figure 2B depicts the nucleotide sequence of the dms A2 promoter (SEQ ID NO: 4). Figure 2C depicts the nucleotide sequence of the dms A3 promoter (SEQ ID NO: 5). Figure 3: Depicts the FNR sequences including the consensus sequence (SEQ ID: 6), the dmsA and dmsA2 promoters (SEQ ID: 7) , the dmsA3 promoter (SEQ ID: 8). and the m>15 promoter (SEQ ID: 9). Underlined are bases matching the consensus. Figure 4: A graphic description of pTETdmsA and its derivatives.

Figure 5A-B: Figure 5 A depicts the Commassie blue stained polyacrylamide gel showing the expression in S.typhi CVD908/ztr-4 of fragments C from plasmids with different promoters. Figure 5B depicts the perspective Western blot stained with anti-fragment C antibodies.

Figure 6A-D: Shows the effect of anaerobiosis and nitrate on the expression of fragment C in S.typhi CVD90ShtrA. Figure 6A - CVD908htrA(pTET-im-.Λ). Figure 6B - CVD908htrsA(pTET-im-.A2). Figure 6C - CVD908htrsA(pTET-imsA3). Figure 6D - CVD908htrsA(pTET«/rl5). DETAILED DESCRIPTION OF THE INVENTION:

The invention described in detail herein involves modified live bacterial vectors capable of expressing protective antigens or therapeutic proteins in an in vitro or in vivo environment. In a preferred embodiment, the vaccine vector delivers the antigens to the immune system, thereby stimulating a protective immune response. The invention specifically involves the targeted delivery and expression of therapeutic proteins to animal host cells using aerobically induced and nitrate inhibited dmsA promoters and derivatives. The promoters described in detail herein allow for the controlled manipulation of protein expression in a variety of environments, including anaerobic and nitrate-rich conditions.

Construction Of The Expression Cassette The particular expression cassette housing the gene or DNA sequence of interest is not critical to the present invention. The cassette is preferably a plasmid and may be selected from any of the many commercially available plasmids. A preferred plasmid of the invention is pTET. The plasmid may be further modified to change the tissue specificity of the invasive bacteria. Likewise, additional genetic elements may be included on the plasmid in order to modify its behavior inside the bacteria or the recipient animal cell. The only critical feature of the plasmid is the operative connection of the DNA sequence of interest to the dmsA promoter of the present invention. Other modifications will be readily appreciated by those skilled in the art.

Therapeutic Genes of Interest The gene of interest may encode an antigenic protein useful as a vaccine. The vaccine antigen of interest may be a protein or antigenic fragment thereof from viral pathogens, bacterial pathogens, parasitic pathogens, and cancer related antigens. Alternatively, the vaccine antigen may be a synthetic gene, constructed using recombinant DNA methods, which encode antigens or parts thereof from viral pathogens, bacterial pathogens, parasitic pathogens, and cancer related antigens. These pathogens can be infectious in humans, domestic animals or wild animal hosts. The antigen can be any molecule that is expressed by any viral, bacterial, parasitic pathogen prior to or during entry into, colonization of, or replication in their animal host.

The viral pathogens, from which the viral antigens are derived, include, but are not limited to, Orthomyxoviruses, such as influenza virus; Retroviruses, such as RSV and SIV, Herpes viruses, such as EBV; CMV or herpes simplex virus; Lentiviruses, such as human immunodeficiency virus; Rhabdoviruses, such as rabies; Picomoviruses, such as poliovirus; Poxviruses, such as vaccinia; Rotavirus; and Parvoviruses. Examples of protective antigens of viral pathogens include the human immunodeficiency virus antigens Nef, p24, gpl20, gp4 i. Tat. Rev, and Pol et al. Nature, 313:277-280 ( 1985)) and T cell and B cell epitopes of gpl20 (Palker et al, f. Immunol. 142:3612-3619 ( 1989)); the hepatitis B surface antigen (Wu et al, Proc. Natl. Acad. Sci., USA, 86:4726-4730 ( 1989)): rotavirus antigens. such as VP4 (Mackow et al, Proc. Natl. Acad. Sci, USA, 87:518-522 ( 1990)) and VP7 (Green et al. J. Virol., 62: 1819-1823 (1988)), influenza virus antigens such as hemagglutinin or nucleoprotein (Robinson et al., Supra; Webster et al. Supra) and herpes simplex virus thymidine kinase (Whitley et al, In: New Generation Vaccines, pages 825-854).

The bacterial pathogens, from which the bacterial antigens are derived, include but are not limited to, Mycobactenum spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coli,

Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio spp., and Borellia burgdorferi.

Examples of protective antigens of bacterial pathogens include the Shigella sonnei form 1 antigen (Formal et al, Infect. Immun., 34:746-750 (1981)); the O-antigen of V. cholerae Inaba strain 569B (Forrest et al, J. Infect. Dis., 159:145-146 (1989); protective antigens of enterotoxigenic E. coli, such as the CFA/I fimbrial antigen (Yamamoto et al, Infect. Immun., 50:925-928 (1985)) and the nontoxic B-subunit of the heat-labile toxin (Clements et al, 46:564-569 ( 1984)); pertactin of Bordetella pertussis (Roberts et al, Vacc, 10:43-48 (1992)), adenylate cyclase-hemolysin of B. pertussis (Guiso et al, Micro. Path. , 1 1 :423-431 (1991)), and fragment C of tetanus toxin of Clostridium tetani (Fairweather et al, Infect. Immun., 58: 1323-1326 ( 1990)).

The parasitic pathogens, from which the parasitic antigens are derived, include but are not limited to, Plasmodium spp., Trypanosome spp., Giardia spp., Boophilus spp., Babesia spp., Entamoeba spp., Eimeria spp., Leishmania spp., Schistosome spp., Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerea spp. Examples of protective antigens of parasitic pathogens include the circumsporozoite antigens of Plasmodium spp. (Sadoff et al, Science, 240:336-337 ( 1988)), such as the circumsporozoite antigen of P. bergerii or the circumsporozoite antigen of P. falcipanim; the merozoite surface antigen of Plasmodium spp. (Spetzler et al. Int. f. Pept. Prot. Res. , 43:351-358 ( 1994)); the galactose specific lectin of Entamoeba histolytica (Mann et al. Proc. Natl. Acad. Sci., USA, 88:3248-3252 ( 1991)), gp63 of Leishmania spp. (Russell et al. J. Immunol. 140: 1274- 1278 ( 1988)), paramyosin of Brugia malayi (Li et al, Mol. Biochem. ParasitoL, 49:315-323 ( 1991 )), the triose-phosphate isomerase of Schistosoma mansoni (Shoemaker et al. Proc. Natl. Acad. Sci., USA, 89: 1842- 1846 ( 1992)); the secreted globin-like protein of Trichostrongylits colubrifonnis (Frenkel et al, Mol. Biochem. ParasitoL, 50:27-36 ( 1992)); the glutathione-S-transferase's of Frasciola hepatica (Hillyer et al. Exp. ParasitoL. 75: 176- 186 (1992)), Schistosoma bovis and S. japonicum (Bashir et al, Trop. Geog. Med., 46:255-258 ( 1994)); and KLH of Schistosoma bovis and S. japonicum (Bashir et al, supra).

The Bacterial Vector As used herein, "bacterial vectors" are defined as bacteria that are capable of delivering foreign antigens to animal cells or animal tissue. "Bacterial vectors" include bacteria that are genetically engineered to deliver foreign antigens to animal tissues.

The particular bacteria employed in the present invention is not critical to the invention. Examples of such bacteria include, but are not limited to Salmonella spp, Shigella spp, Listeria spp., Rickettsia spp., Escherichia spp., Yersinia spp., Klebsiella spp., Bordetella spp., Neissena spp., Aeromonas spp., Franciesella spp., Corynebactenum spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobactenum spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp.. Bacillus spp., and Erysipelothrix spp.

The particular Salmonella strain employed is not critical to the present invention. Examples of Salmonella strains which can be employed in the present invention include Salmonella typhi (ATCC No. 7251) and S. typhimurium (ATCC No. 13311). Attenuated Salmonella strains are preferably used in the present invention and include S. typhi aroAaroD and S. typhimurium aroA mutant. Alteratively, new attenuated Salmonella strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Shigella strain employed is not critical to the present invention. Examples of Shigella strains which can be employed in the present invention include Shigella flexneri 2a (ATCC No. 29903), Shigella sonnei (ATCC No. 29930), and Shigella disenteriae (ATCC No. 13313). An attenuated Shigella strain, such as Shigella flexneri 2a 2457T ΔaroA ΔvirG mutant CVD 1203

(Noriega et al, supra), Shigella flexneri M90T ΔicsA mutant (Goldberg et al, Infect. Immun.,

62:5664-5668 (1994)), Shigella flexneri Y SFL114 aroD mutant (Kamell et al, Vacc, 10: 167-174

(1992)), and Shigella flexneri ΔaroA ΔaroD mutant (Verma et al, Vacc, 9:6-9 (1991)) are

preferably employed in the present invention. Alternatively, new attenuated Shigella spp. strains can be constructed by introducing an attenuating mutation either singularly or in conjunction with one or more additional attenuating mutations.

The particular Listeria strain employed is not critical to the present invention. Examples of Listeria strains which can be employed in the present invention include Listeria monocytogenes

(ATCC No. 15 13). Attenuated Listeria strains, such as L. monocytogenes ΔactA mutant or L.

monocytogenes ΔplcA are preferably used in the present invention. Alternatively, new attenuated

Listeria strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Rickettsia strain employed is not critical to the present invention. Examples of Rickettsia strains which can be employed in the present invention include Ricketsia rickettsiae (ATCC Nos. VR149 and VR891), Ricketsia prowaseckii (ATCC No. VR233), Ricketsia tsutsugamushi (ATCC Nos. VR312, VR150 and VR609), Ricketsia mooseri (ATCC No. VR144), Ricketsia sibirica (ATCC No. VR151), and Rochalimaea quitana (ATCC No. VR358). Attenuated Ricketsia strains are preferably used in the present invention and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Escherichia strain employed is not critical to the present invention. Examples of Escherichia strains which can be employed in the present invention include Escherichia coli strains 4608-58, 1 184-68, 53638-C-17, 13-80, and 6-81 (Sansonetti et al. Ann. Microbiol. (Inst. Pasteur), 132A:351-355 ( 1982)). Other examples include E. coli H10407. and E. coli EFC4, CFT325 and CPZ005. Attenuated Escherichia strains, such as the attenuated turkey pathogen E. coli 02 carAB mutant are preferably used in the present invention. Alternatively, new attenuated Escherichia strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Yersinia strain employed is not critical to the present invention. Examples of Yersinia strains which can be employed in the present invention include Y. enterocolitica (ATCC No. 9610) or Y. pestis (ATCC No. 19428). Attenuated Yersinia strains, such as Y. enterocolitica Ye03-R2 or Y. enterocolitica aroA are preferably used in the present invention. Alternatively, new attenuated Yersinia strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Klebsiella strain employed is not critical to the present invention. Examples of Klebsiella strains which can be employed in the present invention include K. pneumoniae (ATCC No. 13884). Attenuated Klebsiella strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Bordetella strain employed is not critical to the present invention. Examples of Bordetella strains which can be employed in the present invention include R. bronchiseptica (ATCC No. 19395). Attenuated Bordetella strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Neisseria strain employed is not critical to the present invention. Examples of Neisseria strains which can be employed in the present invention include N. meningitidis (ATCC No. 13077) and N. gonorrhoeae (ATCC No. 19424). Attenuated Neisseria strains, such as N. gonorrhoeae MS I 1 aro mutant (Chamberlain et al, Micro. Path.. 15:51-63 ( 1993)) are preferably used in the present invention. Alternatively, new attenuated Neisseria strains can be constnicted by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Aeromonas strain employed is not critical to the present invention. Examples of Aeromonas strains which can be employed in the present invention include A. eucrenophila (ATCC No. 23309). Alternatively, new attenuated Aeromonas strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Franciesella strain employed is not critical to the present invention. Examples of Franiesella strains which can be employed in the present invention include F. tularensis (ATCC No. 15482). Attenuated Franciesella strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Corynebacterium strain employed is not critical to the present invention. Examples of Corynebacterium strains which can be employed in the present invention include C. pseudotiiberculosis (ATCC No. 19410). Attenuated Corynebacterium strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Citrobacter strain employed is not critical to the present invention. Examples of Citrobacter strains which can be employed in the present invention include C. freundii (ATCC No. 8090). Attenuated Citrobacter strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Chlamydia strain employed is not critical to the present invention. Examples of Chlamydia strains which can be employed in the present invention include C. pneumoniae (ATCC No. VR1310). Attenuated Chlamydia strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Hemophilus strain employed is not critical to the present invention. Examples of Hemophilus strains which can be employed in the present invention include H. sornnus (ATCC No. 43625). Attenuated Hemophilus strains are preferably used in the present invention, and can be constmcted by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Brucella strain employed is not critical to the present invention. Examples of Brucella strains which can be employed in the present invention include B. abortus (ATCC No. 23448). Attenuated Brucella strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Mycobactenum strain employed is not critical to the present invention. Examples of. Mycobactenum strains which can be employed in the present invention include M. intracellulare (ATCC No. 13950) and M. tuberculosis (ATCC No. 27294). Attenuated Mycobacterium strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Legionella strain employed is not critical to the present invention. Examples of Legionella strains which can be employed in the present invention include L. pneumophila (ATCC No. 33156). Attenuated Legionella strains, such as a L. pneumophila mip mutant are preferably used in the present invention. Alternatively, new attenuated Legionella strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Rhodococcus strain employed is not critical to the present invention. Examples of Rhodococcus strains which can be employed in the present invention include R. equi (ATCC No. 6939). Attenuated Rhodococcus strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Pseudomonas strain employed is not critical to the present invention. Examples of Pseudomonas strains which can be employed in the present invention include P. aeruginosa (ATCC No. 23267). Attenuated Pseudomonas strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Helicobacter strain employed is not critical to the present invention. Examples of Helicobacter strains which can be employed in the present invention include H. mustelae (ATCC No. 43772). Attenuated Helicobacter strains are preferably used in the present invention, and can be constmcted by introducing one or more attenuating mutations as described for Shigella spp. below. The particular Vibrio strain employed is not critical to the present invention. Examples of

Vibrio strains which can be employed in the present invention include Vibrio cholerae (ATCC No. 14035) and Vibrio cincinnatiensis (ATCC No. 35912). Attenuated Vibrio strains are preferably used in the present invention and include V. cholerae RSI virulence mutant and V. cholerae ctxA, ace, zot, cep mutant. Alternatively, new attenuated Vibrio strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Bacillus strain employed is not critical to the present invention. Examples of Bacillus strains which can be employed in the present invention include Bacillus subtilis (ATCC No. 6051). Attenuated Bacillus strains are preferably used in the present invention and include B. anthracis mutant pXOl and attenuated BCG strains. Alternatively, new attenuated Bacillus strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

The particular Erysipelothrix strain employed is not critical to the present invention. Examples of Erysipelothrix strains which can be employed in the present invention include Erysipelothrix rhusiopathiae (ATCC No. 19414) and Erysipelothrix tonsillarum (ATCC No. 43339). Attenuated Erysipelothrix strains are preferably used in the present invention and include E. rhusiopathiae Kg- la and Kg-2 and E. rhusiopathiae OR VAC mutant. Alternatively, new attenuated Erysipelothrix strains can be constructed by introducing one or more attenuating mutations as described for Shigella spp. below.

Attenuating mutations can be introduced into bacterial pathogens using nonspecific mutagenesis either chemically, using agents such as N-methyl-N'-nitro-N-nitrosoguanidine, or using recombinant DNA techniques; classic genetic techniques, such as TnlO mutagenesis,

P22-mediated transduction, λ phage mediated crossover, and conjugational transfer; or site-directed

mutagenesis using recombinant DNA techniques. Recombinant DNA techniques are preferable since strains constructed by recombinant DNA techniques are far more defined. Examples of such attenuating mutations include, but are not limited to: (a) auxotrophic mutations, such as aro , gua, nad, thy, andasd mutations;

(b) mutations that inactivate global regulatory functions, such as cya, crp, phoPlphoQ, phoP^c or ompR mutations;

(c) mutations that modify the stress response, such as recA, htrA, htpR, hsp and groΕL mutations; (d) mutations in specific virulence factors, such as IsyA, pag or prg iscA or v rG , pic A and act mutations;

(e) mutations that affect DNA topology, such as top A mutation;

(f) mutations that block biogenesis of surface polysaccharides, such as rflb, galE mutations;

(g) mutations that modify suicide systems, such as sacB, nuc, hok, gefl kil, or phi A mutations;

(h) mutations that introduce suicide systems, such as lysogens encoded by P22, λ murein

transglycosylase or S-gene; and

(i) mutations that disrupt or modify the correct cell cycle, such as m/«B mutation. The bacteria of the present invention are generally administered along with a pharmaceutically acceptable carrier or diluent. The particular pharmaceutically acceptable carrier or diluent employed is not critical to the present invention. Examples of diluents include a phosphate buffered saline, buffer for buffering against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone (Levine et al, J. Clin. Invest., 79:888-902 ( 1987); and Black et al J. Infect. Dis., 155: 1260-1265 (1987)), or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame (Levine et al. Lancet, 11:467-470 (1988)). Examples of carriers include proteins, e.g., as found in skim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically these carriers would be used at a concentration of about 0.1-90% (w/v) but preferably at a range of 1-10% (w/v). The amount of the live invasive bacteria of the present invention to be administered will vary depending on the species of the subject, as well as the disease or condition that is being treated and the bacteria being used. For example, the dosage employed with Salmonella will be about 10³ to 10¹¹ viable organisms, preferably about 10⁵ to 10⁹ viable organisms.

The mucosal and systemic immune systems are compartmentalized (Mesteky, J. Clin. Immunol, 7:265-270 (19870; Newby, In: Local Immune Response of the Gut, Boca Raton, CRC Press. Newby and Stocks Eds., pages 143-160 (1984): and Pascual et al., Immuno. Methods., 5:56-72 (1994)). Thus, antigens delivered to mucosal surfaces elicit mucosal and systemic responses, whereas parentally delivered antigens elicit mainly systemic responses but only stimulate poor mucosal responses (Mesteky, supra). Moreover, mucosal stimulation at one mucosal site (for example the intestine) can result in development of immunity at other mucosal surfaces (for example genital/urinary tract) (Mesteky, supra). This phenomenon is referred to as the common mucosal system and is well documented (Mesteky, supra; and Pascual et al, supra).

Successful administration requires the presentation of the antigen to the appropriate cell of the immune system. Preferred administration routes include those that target sites of the immune system, such as the mucosa or the lymph tissues. Thus, intranasal, intraocular, intrarectal, and intravaginal are preferred administration routes.

EXAMPLES The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention. Part I. Materials and methods

Genetic manipulations and cloning procedure. Restriction enzymes and Vent polymerase were from New England Biolabs, Beverly, MA. Primers (Olitogo, Cruachem, Dulles, VA) were designed according to the published sequence of dmsA (3) (Figure 1), and included specific restriction sites for cloning. The fragments were PCRed from the bacterial template with Vent polymerase followed by short extension with Taq polymerase (GIBCO BRL, Gaithersburg. MD). The fragments were then ligated into pGEM-T cloning vector (Promega) and the resulting plasmids were transformed into E. coli DH5 (Gibco BRL, Gaithersburg, MD). Plasmid preparations made from these clones served as a source for the fragments cloned in the final expression plasmids (Table 1). Finally, the expression plasmids were electroporated into CVD908btrA (13) by using BioRad Gene Pulsar II (2.5 Volt, 25μf, 200Ω).

Table 1. Strains and lasmids

Bacterial strains and growth conditions. E. coli DH5 were grown in Luria Base (LB) agar (Gibco BRL, Gaithersburg, MD) supplemented with 50μg / ml carbenicillin (Sigma, St. Louis, MO). S. typhi CVD908btrΛ harboring the different expression plasmids were streaked from frozen stocks onto LB agar supplemented with 0.0001% (w/v) 2.3-dihydroxybenzoic acid (DHB, Sigma. St. Louis, MO) and 50μg / ml carbenicillin. After overnight incubation at 37°C, isolated colonies were inoculated into LB broth containing DHB and carbenicillin, and incubated at 37°C, 250 RPM. For expression under aerobic conditions, late log or stationary phase cultures were inoculated into LB-broth containing DHB and carbenicillin. and incubated as described above. For anaerobic induction, Oxyrase solution (Oxyrase, Mansfield, OH) was added to the broth and the bacteria were incubated static at 37°C. Detection of protein expression. Cultures were grown to a late log phase (O.D.600 = 1.0) and were concentrated 40 folds. About 1.5 X 10⁶ bacteria were loaded on each lane of 10% polyacrylamide gel and proteins were separated by SDS-PAGE in Bio-Rad Mini-PROTEAN II (Bio-Rad, Hercules, CA) and stained with coomassie blue. For Western blots, separated proteins were transferred to an Immunolite membrane (Bio-Rad, Hercules, CA), using Bio-Rad Mini Trans-Blot and immunostained with monoclonal mouse anti-fragment C antibodies (Boehringer Mannheim). Membranes were then incubated with peroxidase labeled goat anti-mouse IgG (Gibco BRL, Gaithersburg, MD) followed by chemiluminisence substrate ECL (Amersham, England). The specific signals were detected by X-ray film (KODAK, Rochester, NY).

Immunization of mice. Balb/C mice, 8-10 weeks of age (Charles River Laboratories) were lightly anaesthetized with fluothane and immunized intranasally as previously described (8). Animals were bled retro-orbitally prior and 14 days following each immunization and sera was collected and kept at -20 until assayed.

Measurement of serum IgG response. Serum antibody response was measured by enzyme-linked immunosorbent assay (ELISA). Polystyrene "U" bottom 96-wells microtiter plates (Dynatech) were coated with 0.2 μg tetanus toxoid (TT) (Connaught Labs, Swiftwear, PA) per well and blocked with 5% fetal bovine serum (FBS) in PBS. Individual serum samples were diluted in PBS containing 0.05% Tween-20 and 1% FBS, and added to the plates in serial two-fold dilutions. A standard positive serum was included in each plate as a control. The plates were then developed with alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Kikegaard and Perry Labs) followed by -nitrophenylphosphate substrate (Kikegaard and Perry Labs). Reactions were terminated with 3N NaOH, and net optical densities were measured at 405 nm. End-point titers were defined as the maximum dilution giving an O.D.450 greater than 0.2. The cut-off was determined as the mean plus two standard deviations above the mean optical density value of 11 naive mouse serum run at a 1 :50 dilution.

Tetanus toxin challenge assay. Immunized mice were challenged with tetanus toxin as previously described (8). Briefly, 100 LD50 of the toxin suspended in 0.5 ml of PBS containing 0.2% gelatin were injected intraperitoneally (i.p.) into the lower right quadrant. Mice were scored for symptoms during a 5-day period after challenge.

Part II. Results Cloning of dmsA promoter and expression in CVD908/ztrA. The published sequence of dmsA (3) was used to determine the fragment containing dms A promoter. The primers 5 'DMS A and 3'DMSA were designed such that they will include an EcoR I and Bgl II sites in the 5' and the 3' ends respectively (Table 2). Table 2. Primers used for PCR.

The fragment containing dmsA from S. typhi and from E. coli DH5 was amplified. Since the results of the reactions were better when using E. coli DH5 as a template, the 512bp fragment containing dmsA from E. coli was cloned into pTETm>15 thus replacing m>15 promoter (Figure 4). The resulting plasmid, pTETdmsA, was electroporated into CVD908btrA and tested for expression of fragment C. The expression of fragment C initiated by dmsA was detectable in western blot by using monoclonal antibodies against fragment C (Figure 5). However, the level of expression was significantly lower then that obtained with pTETπ/rl5. A new 3' primer (3'DMSA2) was designed, which truncated a 167 bp non-translated sequence between transcription initiation point and the starting codon ATG. The resulting promoter. dmsA2, was cloned as described for dmsA, driving the expression of fragment C. The level of expression obtained with pTETdmsA2 was much higher than that obtained with the original dmsA promoter and was controlled by anaerobic growth conditions (Figure 5). The expression was easily detected in commassie-blue stained gels as well as in western blot. However, the level of expression was still lower then that achieved with w>15 promoter. Since the FNR sequence of dmsA and dmsA2 is significantly different from that of the consensus sequence of FNR promoters (Figure 3), another 5' primer, 5'DMSA3 was designed. This primer served two functions: (i) 5'DMSA3 optimized the FNR sequence according to the consensus FNR sequence and (ii) it truncated a 264 bp upstream sequence which, according to the FNR model, is not essential for the transcriptional control of dmsA. The resulting promoter, dmsA3, was cloned as described earlier and the plasmid X \ TEYdmsA3 was electroporated into CVD908/ztrA. As shown in Figure 6, the level of expression obtained by fYΕldmsA3 is comparable to that obtained with pTEtnirlS. The expression was tightly controlled by anaerobic growth conditions and could be easily detected in commassie blue stained SDS-PAGE gels as well as in Western blot. Response to nitrate. The original dmsA promoter contains narL responsive elements and is repressed by nitrate. Thus, it was interesting to test the effect of nitrate in combination with anaerobic growth conditions on the level of expression of fragment C. The addition of nitrate did not seem to have any effect on the very low level of expression detected under aerobic conditions (Figure 6). Under anaerobic conditions however, the addition of nitrate repressed to some extent the expression controlled by dmsA and dmsA2. A nitrate effect on the expression controlled by dmsA3 and nir!5 could not be detected. Immunogenicitv in mice. Mice were immunized with two doses of CVD908/-t harboring the expression plasmids pTETJm_?A2 or pTETdmsA3. Two control groups were immunized with the vaccine strain harboring pTETm>15 and with the vaccine strain alone. Senim samples obtained in two independent trials for antibodies against tetanus toxin and S. typhi LPS were analyzed by ELISA. The results (Table 3), show that CVD908btrA(pTETαfm.sAi) generated the highest level of anti-fragment C antibodies with geometric mean titer (GMT) of 55834. CVD908/ztrA(pTETm^'rl5) elicited a significantly lower response with GMT of 4354 (p=0.007). CVO 0BhtrA(pTETώnsA2) generated very low anti-tetanus response that was not significantly higher then that of the control group that was immunized with the carrier strain alone (p=0.23) and was significantly lower then that obtained with the bacteria carrying pTETm^'rlS or pTETdmsA3 (p<0.001 and p<0.001 respectively). All of the groups gave the same levels of anti-5. typhi LPS antibodies. Mice were challenged with 100 LD50 of tetanus toxin injected i.p. All naive animals died within 24 hours after the challenge. Immunization with CVD908/ztr (pTET dmsA2) gave only marginal protection and six out of the seven mice died between 24 to 48 hours after challenge. The only surviving mouse was the one that developed the highest titer of anti-tetanus toxin antibodies. Three mice that were immunized with CVD908btrA(pTET-ϊ^/m& 5) were challenged and all survived. Four mice immunized with CVD908/ztrA(pTETm^'rl5) were challenged. Three mice survived the challenge while one was partially paralyzed 24 hours after challenge and was found dead 48 hours after challenge.

Table 3. IgG antibody response in mice sera after two doses of 5. typhi CVO90ShtrA expressing fragment C under the control of different promoters.

aNumber of animals analyzed in each group, t vs <J, p<0.001 ; c vs e, p=0.007; d vs e, p<0.001 N vs b, p=0.23. Part III. Discussion

In the example discussed above, the use of the anaerobic promoter dmsA to control the expression of a foreign gene, fragment C of tetanus toxin, in the attenuated S. typhi CVD908btrA vaccine strain was studied. For the cloning, the relevant primers were designed according to the published sequence derived form E. coli. The cloned dmsA drove the expression of fragment C under anaerobic growth conditions and the response under anaerobic conditions was repressed by nitrate. It has been suggested that nitrate regulation via NarL controls the expression during both anaerobic and aerobic growth conditions (11). In the example described in detail herein, nitrate repression under aerobic conditions could not be detected. This result may result may be due to the low residual activity during aerobic conditions combined with the relatively low sensitivity of the western blot.

In recent work, it was shown that the non-translated sequence down stream of the transcription initiation point includes two regulatory elements, IHF and ModΕ binding sequences (16). In that work, they showed that ModΕ sequence is required for regulation of dmsA-lacZ fusion expression in response to oxygen and nitrate. Specifically, they showed that deleting this sequence led to a lower level of a reporter gene. In the examples of the invention described herein, the promoter derivative dmsAl, a product of a similar deletion, yielded a significant increase of fragment C expression under anaerobic conditions. Though net wishing to be bound by theory, it is proposed that the differences between the two studies can be explained by either differences in specific sequences in the final gene fusion that can lead to differences in mRNA stability or differences in the background strain.

To further improve the level of expression, the 5' upstream sequence was truncated and the FNR binding sequence was optimized. According to the general FNR model, the upstream sequence to the FNR site is not essential for the FNR and nitrate regulation. The results herein show that, indeed, the level of expression induced by dmsA3 was higher then that of dmsA2 and was at the same level as that induced by mV15. This result reflects the similarity in the FNR sequences of dmsA3 and m^'rl5. Surprisingly, in the case of dmsA3, the repression effect of nitrate under anaerobic growth conditions could not be detected.

In a recent study, it was shown that the upstream sequence of nirB promoter contains specific binding sites for proteins that regulate the effect of NarL on FNR (20). Although in the case of nirB, NarL work in concert with FNR to enhance the activity of the promoter, it is possible that similar interaction regulate the inhibitory effect of NarL in dmsA promoter. However, the inability to show the inhibitory effect of nitrate in the case of dmsA3 may be a result of the low sensitivity of the assay together with the very high expression level induced by the optimized FNR sequence that could mask the limited effect of nitrate.

The results obtained with mice immunized intranasally with the attenuated vaccine strains indicated that pTETdmsA3 was more efficient then pTETdmsA2 in eliciting anti-fragC IgG response. This is not surprising in light of the differences in the level of expression derived from dmsA3 and dmsA2. This result is further supported by the challenge study where only one out of seven mice immunized with CVD908btrA(pTET-tm& 2) was protected. Surprisingly, pTET-imsAi exceeded pTETrnV15 in inducing anti-fragC antibodies. The results obtained with pTETdmsA2 suggested that the constitutive low level activity of the promoters under aerobic growth conditions did not lead to a significant antibody response. Thus, while not wishing to be bound by theory, it is presumed that there must be some level of induction of the promoter in vivo in order to get immune response against fragment C. On the other hand, there were no major differences in the level of expression induced in vitro by CVD908 ztrA carrying pTET<fm.sA3 or pTETrn^'rl5. It is speculated that the differences in the immune response might reflect differences in the induction in vivo of the two promoters. In examples described herein, dmsA promoter derivatives are used for expression of foreign antigens in live attenuated vaccine. The promoters were able to drive the expression of a model protein, fragment C of tetanus toxin. One derivative, dmsA3, was able to elicit protective immune response in mice against challenge with the toxin. The use of dmsA derivatives in live attenuated vaccines offers a modular promoter system for expression of specific genes at different levels and in a controlled manner. This allows tailoring an optimal expression system by matching the level of expression of a specific gene to the carrier strain. All references cited herein are incorporated by reference in their entirety.

(1) Anderson, R., G. Dougan, and M. Roberts. 1996. Delivery of the Pertactin/P.69 polypeptide of Bordetella pertussis using an attenuated Salmonella typhimurium vaccine strain: expression levels and immune response. Vaccine. 14:1384-90.

(2) Barry, E. M., O. Gomez-Duarte, S. Chatfield, R. Rappuoli, M. Pizza, G. Losonsky, J. Galen, and M. M. Levine. 1996. Expression and immunogenicity of pertussis toxin SI subunit-tetanus toxin fragment C fusions in Salmonella typhi vaccine strain CVD 908. Infect Immun. 64:4172-81.

(3) Bilous, P. T., S. T. Cole, W. F. Anderson, and J. H. Weiner. 1988. Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli. Mol Microbiol. 2:785-95.

(4) Chatfield, S. N., I. G. Charles, A. J. Makoff, M. D. Oxer, G. Dougan, D. Pickard, D. Slater, and N. F. Fairweather. 1992. Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine. Biotechnology (N Y). 10:888-92.

(5) Cotter, P. A., and R. P. Gunsalus. 1989. Oxygen, nitrate, and molybdenum regulation of dmsABC gene expression in Escherichia coli. J Bacteriol. 171:3817-23.

(6) Darwin. A. J., and V. Stewart. 1996. The NAR modulon systems: Nitrate and nitrite regulation of anaerobic gene expression. Coordinating eds., E. C. C. Lin, and S. A. Lynch. R. G. Landes

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(7) Everest, P., G. Frankel, J. Li, P. Lund, S. Chatfield, and G. Dougan. 1995. Expression of LacZ from the htrA. nirB and groE promoters in a Salmonella vaccine strain: influence of growth in mammalian ceils, f EMS Microbiol Lett. 126:97-101.

(8) Galen, J. E., O. G. Gomez-Duarte, G. A. Losonsky, J. L. Halpern, C. S. Lauderbaugh, S. Kaintuck. M. K. Reymann, and M. M. Levine. 1997. A murine model of intranasal immunization to assess the immunogenicity of attenuated Salmonella typhi live vector vaccines in stimulating serum antibody responses to expressed foreign antigens. Vaccine. 15:700-8.

(9) Gomez-Duarte, O. G., J. Galen, S. N. Chatfield, R. Rappuoli, L. Eidels, and M. M. Levine. 1995. Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain. Vaccine. 13: 1596-602.

(10) Guest. J. R., J. Green, A. S. Irvin, and S. Spiro. 1996. The FNR modulon and FNR-regulated gene expression. Coordinating eds.. E. C. C. Lin. and S. A. Lynch. R. G. Landes Company.

( 1 1) Gunsalus, R. P. 1992. Control of electron flow in Escherichia coli: coordinated transcription of respiratory pathway genes. J Bacteriol. 174:7069-74.

(12) Jayaraman, P. S., T. C. Peakman, S. J. Busby. R. V. Quincey, and J. A. Cole. 1987. Location and sequence of the promoter of the gene for the NADH- dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite. J Mol Biol. 196:781-8.

(13) Levine, M. M., J. Galen, E. Barry, F. Noriega. S. Chatfield, M. Sztein, G. Dougan. and C. Tacket. 1996. Attenuated Salmonella as live oral vaccines against typhoid fever and as live vectors. J Biotechnol. 44: 193-6.

(14) Londono, L. P., S. Chatfield, R. W. Tindle, K. Herd. X. M. Gao, I. Frazer, and G. Dougan. 1996. Immunisation of mice using Salmonella typhimurium expressing human papillomavirus type 16 E7 epitopes inserted into hepatitis B virus core antigen. Vaccine. 14:545-52.

(15) Lynch, A. S., and E. C. C. Lin. 1996. Responses to molecular oxygen, p. 1526-48. In F.C. Neidhart (ed.), Escherichia coli and Salmonella typhimirum, 2nd ed, vol. 1. ASM Press, Washington, DC. (16) McNicholas, P. M., R. C. Chiang, and R. P. Gunsalus. 1998. Anaerobic regulation of the Escherichia coli dmsABC operon requires the molybdate-responsive regulator ModE. Mol Microbiol. 27: 197-208.

(17) Oxer, M. D., C. M. Bentley, J. G. Doyle, T. C. Peakman, I. G. Charles, and A. J. Makoff. 1991. High level heterologous expression in E. coli using the anaerobically- activated nirB promoter. Nucleic Acids Res. 19:2889-92.

(18) Tseng, C. P., J. Albrecht, and R. P. Gunsalus. 1996. Effect of microaerophilic cell growth conditions on expression of the aerobic (cyoABCDE and cydAB) and anaerobic (narGHJI, frdABCD, and dmsABC) respiratory pathway genes in Escherichia coli. J Bacteriol. 178: 1094-8.

(19) Tseng, C. P., A. K. Hansen, P. Cotter, and R. P. Gunsalus. 1994. Effect of cell growth rate on expression of the anaerobic respiratory pathway operons frdABCD, dmsABC, and narGHJI of Escherichia coli. J Bacteriol. 176:6599-605.

(20) Wu, H., K. L. Tyson, J. A. Cole, and S. J. Busby. 1998. Regulation of transcription initiation at the Escherichia coli nir operon promoter: a new mechanism to account for co-dependence on two transcription factors. Mol Microbiol. 27:493-505.

While the invention has been described in detail, and with reference to specific embodiments thereof, it will be apparent to one with ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

CLAIMS: I, the inventor claim:

1. A method for inducing a therapeutic response in vivo in an animal host comprising administering to the host a live bacterium which contains an anaerobically inducible prokaryotic promoter selected from the group consisting of dmsA, dmsA2, dmsA3 or derivatives thereof operably linked to a DNA sequence encoding a therapeutic protein.

2. A method of claim 1 wherein said therapeutic response is an immune response and said therapeutic protein is a vaccine antigen.

3. The method of claim 2 wherein said immune response involves the production of antibodies at levels sufficient to confer immunity to the host against said vaccine antigen.

4. The method of claim 1 wherein said animal host is a mammal.

5. The method of claim 4 wherein said mammal is selected from the group consisting of humans, bovines, ovines, porcines, felines, buffalos, canines, goats, equines, donkeys, deer, and primates.

6. The method of claim 4 wherein said mammal is a human.

7. The method of claim 1 wherein said bacterium is administered to a mucosal surface of said host.

8. The method of claim 7 wherein said mucosal surface is selected from the group consisting of nasal, ocular, intestinal, rectal, and vaginal tissues.

9. The method of claim 1 wherein said bacterium is intranasally administered.

10. The method of claim 1 wherein said bacterium is orally administered.

11. The method of claim 1 wherein said bacterium selected from the group consisting of Shigella spp., Listeria spp., Rickettsia spp. and Escherichia coli.

12. The method of claim 1 wherein said bacterium is Salmonella spp.

13. The method of claim 1 wherein said bacterium is Salmonella typhimurium.

14. The method of claim 1 wherein said bacterium is selected from the group consisting of Yersinia spp.. Escherichia spp.. Klebsiella spp., Bordetella spp., Neisseria spp.. Aeromonas spp.. Franciesella spp. , Corynebacterium spp. , Citrobacter spp. , Chlamydia spp. , Hemophilus spp.. Brucella spp. , Mycobacte um spp. , Legionella spp. , Rhodococcus spp.. Pseudomonas spp. , Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp.

15. The method of claim 1 wherein said bacterium is attenuated.

16. The method of claim 15 wherein said attenuated bacterium is Salmonella spp.

17. The method of claim 15 wherein said attenuated bacterium is Salmonella typhimurium.

18. The method of claim 15 wherein said attenuated bacterium is S. typhi CVD908/ztrA.

19. A therapeutic vector for administration to an animal host comprising a pharmaceutically effective carrier or diluent, a live bacterium which contains an anaerobically inducible prokaryotic promoter selected from the group consisting of dmsA. dmsA2, dmsA3 or derivatives thereof operably linked to a DNA sequence encoding a therapeutic protein.

20. The vector of claim 20 wherein said therapeutic protein is a vaccine antigen and said therapeutic vector is a vaccine.

21. The vector of claim 20 wherein said bacterium is attenuated.

22. The vector of claim 19 wherein said bacterium selected from the group consisting of Shigella spp., Listeria spp., Rickettsia spp. and Escherichia coli.

23. The vector of claim 19 wherein said bacterium is Salmonella spp.

24. The vector of claim 23 wherein said bacterium is Salmonella typhimurium.

25. The vector of claim 21 wherein said attenuated bacterium is Salmonella spp.

26. The vector of claim 21 wherein said attenuated bacterium is Salmonella typhimurium.

27. The vector of claim 21 wherein said attenuated bacterium is S. typhi CVD908/ιtrA.

28. The vector of claim 19 wherein said bacterium is selected from the group consisting of Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobactenum spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp.

29. A recombinant DNA promotor comprising the consensus sequence

A AAATTTGATAT ATATCAAAT T T

30. An independently functioning expression cassette comprising an origin of replication; a promoter wherein the expression of said promoter is anaerobically induceable; and the nucleic acid sequence of a desired product wherein the transcription of said nucleic acid sequence is controlled by said promoter.

31. An independently functioning expression cassette of Claim 30 wherein said promoter contains the consensus sequence

A AAATTTGATAT ATATCAAAT T T

32. An amplifiable plasmid replicon comprising the independently functioning expression cassette of Claim 30.

33. A bacterial cell comprising the amplifiable plasmid replicon of Claim 32.

34. The amplifiable plasmid replicon of Claim 32 wherein said promoter is the dmsA2 promoter.

35. The amplifiable plasmid replicon of Claim 32 wherein said promoter is the dmsA3 promoter.

36. The amplifiable plasmid replicon of Claim 32 wherein said promoter is a modified dmsA promoter characterized by exhibiting higher rates of anaerobically induced expression in relation to a corresponding dmsA promoter without such mutations.