# US 8,465,755 B2 · Recombinant bacterium capable of eliciting an immune response against enteric pathogens > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,465,755 B2 - Document ID: US8465755B2 - Status: Granted - Kind: B2 - Application number: 12/681,721 - Filed: 2008-10-06 - Published: 2011-02-10 - Granted: 2013-06-18 - Earliest claimed: 2007-10-05 - Assignees: Arizona Board of Regents for and on behalf of Arizona State University - Applicants: Roy Curtiss, III; Melha Mellata; Bereket Zekarias; Zhaoxing Shi; Christine Branger; Kenneth Roland - Inventors: Roy Curtiss, III; Melha Mellata; Bereket Zekarias; Zhaoxing Shi; Christine Branger; Kenneth Roland - Web page: https://patentgrep.com/patent/US8465755B2 ## CPC - A61K 39/0275: {Salmonella} - A61K 39/025: {Enterobacteriales, e.g. Enterobacter} - A61P 37/04: Immunostimulants - C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora - A61K 2039/522: {avirulent or attenuated} - A61K 2039/523: {expressing foreign proteins} - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The present invention relates to a recombinant bacterium that is capable of eliciting an immune response against at least two enteric pathogens, without substantially inducing an immune response specific to the serotype of the bacterium. The invention also relates to methods of making such a bacterium and vaccines and methods of using such a bacterium. ## Claims ### Claim 1 A recombinant Salmonella bacterium, wherein (a) the recombinant bacterium is selected from the group consisting of S. Typhimurium, S. Paratyphi, or S. Typhi, and comprises one or more mutations that reduce serotype-specific immune responses caused by at least one serotype-specific antigen selected from LPS O-antigen, a component of a flagellum, or the Vi capsular antigen, wherein the one or more mutations are selected from the group consisting of: (i) a pmi mutation that results in lack of function of the pmi gene product, (ii) a rfc mutation, such that LPS O-antigen synthesis is greater during in vitro growth compared to in vivo growth of the recombinant bacterium, (iii) a galE mutation that results in lack of function of the galE gene product, (iv) a fliB mutation that reduces expression of the structural gene for Phase II flagellar antigen, (v) a fliC mutation that reduces expression of the antigenically variable serotype-specific domain of the Phase I FliC flagellar antigen, and (vi) or a Vi capsular antigen mutation that reduces synthesis of the Vi capsular antigen; (b) the recombinant bacterium expresses at least one nucleic acid encoding at least two enteric antigens, wherein the enteric antigens are selected from the groups consisting of: (i) Salmonella enterica iron regulated outer membrane protein (IROMP) or manganese regulated outer membrane protein (MnROMP), (ii) Yersinia entercolitica V antigen or psn nucleic acid product, (iii) Shigella species IpaD or aerobactin, (iv) E. coli salmochelin, aerobactin, sit operon nucleic acid product, vaqZ nucleic acid product, a fimbrial operon nucleic acid product, tsh nucleic acid product, iss nucleic acid product, or LTB, (v) C. jejuni PilA or CjaA, and (vi) C. perfringens alpha-toxin or NetB, such that the recombinant bacterium, when administered to a host, is capable of eliciting an immune response against at least two enteric pathogens; and (c) the recombinant bacterium comprises at least one mutation in a nucleic acid regulating the expression of a nucleic acid encoding an IROMP or MnROMP, such that expression of IROMP or MnROMP is greater during in vivo growth of the bacterium compared to in vitro growth of the bacterium. ### Claim 2 (depends on 1) The recombinant bacterium of claim 1, wherein the serotype-specific antigen is LPS O-antigen. ### Claim 3 (depends on 1) The recombinant bacterium of claim 1, wherein the recombinant bacterium comprises the mutation Δpmi-2426. ### Claim 4 (depends on 1) The recombinant bacterium of claim 1, wherein the serotype-specific antigen is a component of a flagellum. ### Claim 5 (depends on 1) The recombinant bacterium of claim 1, wherein the serotype-specific antigen is produced by fliB or fliC. ### Claim 6 (depends on 5) The recombinant bacterium of claim 5, wherein the recombinant bacterium comprises the ΔfljB217 and/or the ΔfliC180 mutation. ### Claim 7 (depends on 1) The recombinant bacterium of claim 1, wherein the recombinant bacterium comprises the mutation ΔPrfc::TT araC PBADrfc. ### Claim 8 (depends on 1) The recombinant bacterium of claim 1, wherein (a) the recombinant bacterium further comprises at least one chromosomally integrated nucleic acid sequence encoding a repressor operably linked to a regulatable promoter, wherein the repressor is selected from the group consisting of LacI, C2, and C1, and the regulatable promoter is selected from the group consisting of PBAD, a promoter regulated by maltose, a promoter regulated by rhamnose, and a promoter regulated by xylose; and (b) the at least one nucleic acid sequence encoding at least two enteric antigens expressed by the recombinant bacterium is operably linked to a promoter regulated by the repressor in (a), such that the expression of the nucleic acid sequence encoding the enteric antigens is repressed during in vitro growth of the recombinant bacterium, but the recombinant bacterium is capable of high level expression of the nucleic acid sequence encoding the enteric antigen in vivo. ### Claim 9 (depends on 8) The recombinant bacterium of claim 8, wherein the recombinant bacterium comprises at least two chromosomally integrated nucleic acid sequences, each encoding a repressor operably linked to a regulatable promoter. ### Claim 10 (depends on 9) The recombinant bacterium of claim 9, wherein each repressor is independently selected from the group consisting of LacI, C2, and C1. ### Claim 11 (depends on 8) The recombinant bacterium of claim 8, wherein the regulatable promoter is PBAD, or a promoter regulated by maltose, rhamnose, or xylose. ### Claim 12 (depends on 8) The recombinant bacterium of claim 8, wherein the bacterium further comprises the mutation ΔPcrp::TT araC PBAD crp. ### Claim 13 (depends on 8) The recombinant bacterium of claim 8, wherein the codons of the nucleic acid sequence encoding the repressor have been modified so as to optimize the expression level of the nucleic acid sequence encoding the repressor. ### Claim 14 (depends on 8) The recombinant bacterium of claim 8, wherein the repressor comprises a modified Shine-Dalgarno sequence and optimized codons so as to optimize the expression level of the nucleic acid sequence encoding the repressor. ### Claim 15 (depends on 8) The recombinant bacterium of claim 8, wherein the nucleic acid encoding an enteric antigen is operably linked to a promoter regulated by a repressor selected from the group consisting of LacI, C1, and C2. ### Claim 16 (depends on 8) The recombinant bacterium of claim 8, wherein the nucleic acid encoding an antigen of interest is operably linked to a Ptrc promoter. ### Claim 17 (depends on 1) The recombinant bacterium of claim 1, wherein the bacterium further comprises the mutation ΔPcrp::TT araC PBAD crp. ### Claim 18 (depends on 1) The recombinant Salmonella bacterium of claim 1, the bacterium comprising a Δpmi mutation, a ΔPrfc::TT araC PBAD rfc mutation, and a ΔgalE mutation. ### Claim 19 (depends on 1) The recombinant Salmonella bacterium of claim 1, the bacterium comprising a ΔPfur::TT araC PBAD fur mutation, a ΔPmntR::TT araC PBAD mntR mutation and a ΔPcrp::TT araC PBAD crp deletion-insertion mutation. ### Claim 20 (depends on 1) The recombinant Salmonella bacterium of claim 1, the bacterium comprising the pAPEC-1 plasmid with a ΔcvaAB::psn iutA iucABCD insertion. ### Claim 21 (depends on 1) The recombinant Salmonella bacterium of claim 1, the bacterium comprising a Δpmi mutation, a ΔPrfc::TT araC PBAD rfc mutation, a ΔgalE mutation, a ΔPfur::TT araC PBAD fur deletion-insertion mutation, a ΔPmntR::TT araC PBAD mntR deletion-insertion mutation and a ΔPcrp::TT araC PBAD crp deletion-insertion mutation, and containing the pAPEC-1 plasmid with the ΔcvaAB::psn iutA iucABCD insertion. ### Claim 22 (depends on 8) The recombinant bacterium of claim 8, wherein the at least one nucleic acid sequence encoding at least two enteric antigens expressed by the recombinant bacterium and operably linked to a promoter regulated by the repressor is on a vector. ### Claim 23 (depends on 22) The recombinant bacterium of claim 22, wherein the vector is a plasmid. ### Claim 24 (depends on 22) The recombinant bacterium of claim 22, wherein the vector further comprises a nucleic acid sequence encoding a secretion signal for the antigen of interest. ### Claim 25 (depends on 22) The recombinant bacterium of claim 22, wherein the repressor is selected from the group consisting of LacI, C2, and C1; the regulatable promoter is PBAD; the vector is a plasmid; and the nucleic acid encoding an enteric antigen is operably linked to the Ptrc promoter. ### Claim 26 (depends on 1) A vaccine composition, the composition comprising a recombinant Salmonella bacterium of claim 1. ### Claim 27 A recombinant Salmonella bacterium, wherein (a) the recombinant bacterium is from the group consisting of S. Typhimurium, S. Paratyphi, and S. Typhi, and comprises one or more mutations that reduce serotype-specific immune responses caused by at least one serotype-specific antigen selected from LPS O-antigen, a component of a flagellum, or the Vi capsular antigen, wherein the one or more mutations are selected from the group consisting of: (i) a pmi mutation that results in lack of function of the pmi gene product, (ii) a rfc mutation, such that LPS O-antigen synthesis is greater during in vitro growth compared to in vivo growth of the recombinant bacterium, (iii) a galE mutation that results in lack of function of the galE gene product, (iv) a fliB mutation that reduces expression of the structural gene for Phase II flagellar antigen, (v) a fliC mutation that reduces expression of the antigenically variable serotype-specific domain of the Phase I FliC flagellar antigen, and (vi) or a Vi capsular antigen mutation that reduces synthesis of the Vi capsular antigen; (b) the recombinant bacterium comprises the mutation ΔPcrp::TT araC PBAD crp; and (c) the recombinant bacterium comprises at least one mutation in fur or mntR. ### Claim 28 A recombinant Salmonella bacterium, wherein (a) the recombinant bacterium is derived-selected from the group consisting of S. Typhimurium, S. Paratyphi, or S. Typhi, and comprises one or more mutations that reduce serotype-specific immune responses caused by at least one serotype-specific antigen selected from LPS O-antigen, a component of a flagellum, or the Vi capsular antigen, wherein the one or more mutations are selected from the group consisting of: (i) a pmi mutation that results in lack of function of the pmi gene product, (ii) a rfc mutation, such that LPS O-antigen synthesis is greater during in vitro growth compared to in vivo growth of the recombinant bacterium, (iii) a galE mutation that results in lack of function of the galE gene product, (iv) a fliB mutation that reduces expression of the structural gene for Phase II flagellar antigen, (v) a fliC mutation that reduces expression of the antigenically variable serotype-specific domain of the Phase I FliC flagellar antigen, and (vi) or a Vi capsular antigen mutation that reduces synthesis of the Vi capsular antigen; (b) the recombinant bacterium comprises the mutation ΔPcrp::TT araC PBAD Crp; (c) the recombinant bacterium comprises at least one mutation in fur or mntR; and (d) the recombinant bacterium expresses at least one enteric antigen selected from the group consisting of (i) Salmonella enterica IROMP or MnROMP, (ii) Yersinia entercolitica V antigen nucleic acid product or psn nucleic acid product, (iii) Shigella species IpaD or aerobactin, (iv) E. coli salmochelin, aerobactin, sit operon nucleic acid product, yagZ nucleic acid product, a fimbrial operon nucleic acid product, tsh nucleic acid product, iss nucleic acid product, or LTB, (v) C. jejuni PilA or CjaA, and (vi) C. perfringens alpha-toxin or NetB.