WO1991018092A1 - Bacille salmonella typhi tolerant les acides, mutants aro doubles de ce bacille et utilisation de ce bacille comme vaccin administre par voie buccale contre la fievre typhoide - Google Patents

Bacille salmonella typhi tolerant les acides, mutants aro doubles de ce bacille et utilisation de ce bacille comme vaccin administre par voie buccale contre la fievre typhoide Download PDF

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WO1991018092A1
WO1991018092A1 PCT/US1991/003447 US9103447W WO9118092A1 WO 1991018092 A1 WO1991018092 A1 WO 1991018092A1 US 9103447 W US9103447 W US 9103447W WO 9118092 A1 WO9118092 A1 WO 9118092A1
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antigen
vaccine
strain
acid
tolerant
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PCT/US1991/003447
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David M. Hone
Myron M. Levine
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University Of Maryland At Baltimore
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    • 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
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to acid-tolerant Salmonella typhi and to double aro mutants of Salmonella typhi which are useful for preparing a single dose oral vaccine against typhoid fever and as a carrier of genes which express protective antigens cloned from other pathogens.
  • the present invention also relates to a method of enrichment of acid-tolerant Salmonella typhi.
  • Salmonella typhi I. Salmonella typhi
  • Salmonella typhi the causative agent of typhoid fever, is a highly host-adapted microorganism which is only fully pathogenic for humans.
  • Typhoid fever is a disease of the reticuloendothelial system.
  • the clinical manifestations of acute typhoid fever usually begin with abdominal discomfort, malaise, fever and headache that increase in a step-wise fashion.
  • Bronchitic cough, nausea, abdominal pains, splenomegaly, rose spots (rare) and leukopenia are also associated with clinical typhoid fever although the frequencies of these signs and symptoms vary. Without early antibiotic intervention, S.
  • typhi infection of humans can lead to intestinal hemorrhage or to the development of a perforated small intestine, which results in life threatening peritonitis.
  • the antibiotics of choice for the treatment of typhoid fever are amoxicillin, trimethoprim/sulphamethozole, chloramphenicol, or ciprofloxacin.
  • mice mice typhoid
  • calves mice typhoid
  • typhoid fever was defined as clinical illness manifested by a temperature of 103°F for at least 36 hours. Many other volunteers in these studies had milder illness accompanied by positive cultures. This was considered typhoid infection but not typhoid fever.
  • the Quailes strain is sensitive to the effects of low pH. Hence, it is quite probable that the pH sensitive characteristic of the Quailes strain might have reduced its capacity to cause disease because of reduced survival through the gastric environment of the stomach and reduced survival in the acidified phagolysosome.
  • S. typhi unlike Salmonella typhimurium which has a much broader host range (fully pathogenic for mice, guinea, pigs, chickens, calves, pigs, and rabbits), is virulent only for humans (Carter et al, Infect. Immun., 10:816-822 (1984)). Following ingestion of virulent S. typhi by the human host, the bacilli reach the small intestine where they invade through the epithelial surface (Takeuchi, Curr. TOP. Pathol., 54:1-27 (1971)).
  • the typhoid bacilli then enter the lymph circulation, spill into the blood circulation via the thoracic duct and cause a silent primary bacteremia (Gaines et al, J. Infect. Dis., 118:293-306 (1968)).
  • the uptake of the typhoid bacilli by fixed macrophages of the reticuloendothelial system during the primary bacteremia leads to dissemination within the liver, spleen, bone marrow and lymph nodes (Gaines et al, J. Infect. Dis., 118:293-306 (1968)).
  • the typhoid bacilli are localized in phagosomes within the macrophages where they remain viable and slowly proliferate.
  • the bacteria emerge from the macrophages of the reticuloendothelial system and give rise to a secondary bacteremia that is accompanied by the onset of clinical symptoms (Hornick et al, N. Engl. J. Med., 283:686-691 (1970)).
  • Typhoid fever currently results in 500,000 deaths each year and causes a great deal more morbidity (Institute of
  • the World Health Organization has targeted the improvement of vaccines against typhoid fever as a priority (World Health Organization Publication
  • Live oral typhoid fever vaccines have been shown to elicit a broad spectrum of cellular and humoral immune responses (Tagliabue et al, Clin. Exp. Immunol., 62:242-247 (1985); Murphy et al, J. Infect. Dis., 156: 1005-1009 (1987); Sarasombath et al, J. Clin. Microbiol., 25:1088-1093 (1987); and Bartholomeusz et al, J. Gastroenterol. Hapatol., 1:61-67 (1987)).
  • Such hybrid live oral vaccine strains expressing foreign protective antigens would be expected to elicit immune responses against the cloned foreign antigens thereby providing protection against the pathogens from which the foreign antigens originated.
  • a live oral typhoid fever vaccine carrying foreign antigens has the potential of very broad applications.
  • Live oral typhoid fever vaccines provide higher levels of protection against challenge with wild-type S. typhi than those achieved by parenteral inactivated vaccines if both humoral as well as effective cell-mediated immune responses are elicited during vaccination.
  • S. typhi In order for S. typhi to stimulate effective cell-mediated immunity, the typhoid bacilli must grow and survive for extended periods within macrophages, whereupon S. typhi antigens are processed and expressed on the surface of the macrophage in association with major histocompatibility antigens HLA-A, B, and C (MHC I) or HLA-DP, DQ, and PR (MHC II).
  • the modified structure of the processed antigen in association with MHC I stimulates CD8 + T-lymphocytes. Further, the modified structure of the processed antigen in association with MHC II stimulates CD4 + T-lymphocytes. Such stimulated T-lymphocytes become activated and it is these activated T-lymphocytes that effect the cell-mediated immune response.
  • Live oral vaccines are at present the only means known by which effective numbers of activated T-lymphocytes, and hence effective cell-mediated immunity, can be elicited.
  • Live oral typhoid fever vaccines if fully attenuated, will have the added advantage of being much less reactogenic than the parenteral typhoid fever vaccines tested by the World Health Organization in the 1960s.
  • the candidate live oral typhoid fever vaccine strains which have heretofore been tested in clinical studies in man include the: (i) streptomycin-dependent S. typhi, strain 27V
  • strains are disadvantageous in that they possesses one or more of the following: insufficient attenuation, poor immunogenicity, the requirement to be administered in multiple doses to achieve serological responses and protection, poor yield after lyophilization, loss of potency following lyophilization, and a lack of precise knowledge of the genetic alterations responsible for attenuation.
  • streptomycin-dependent S. typhi vaccine strain 27V is well-tolerated when given in doses of greater than 10 10 organisms with bicarbonate buffer. However, administration of multiple doses is required to stimulate protective immunity (Levine et al, J.
  • Ty21a includes the necessity to administer multiple doses to achieve a moderate level of protection, a high loss of viability following lyophilization and a lack of precise knowledge about the mutations responsible for attenuation.
  • c. Recombinant Vi + Mutant of Ty21a Strain Ty21a has been modified by recombinant DNA techniques so as to restore the Vi polysaccharide antigen, giving rise to strain Ty21a-Vi + (Cryz et al, Infect. Immun., 57:3863-3868 (1990)). While strain Ty21a-Vi + remained well-tolerated, this strain is not highly immunogenic after ingestion of a single dose or three doses given every other day. d. Recombinant galE Vi- Mutant
  • S. typhi vaccine strain 541Ty harbors a deletion in the aroA gene as well as a deletion in the purA gene (Edwards et al, J. Bacteriol., 170:3991-3995
  • Strain 543Ty is a variant of 541Ty which lacks the Vi polysaccharide antigen that usually covers S. typhi.
  • phage P22 usually transfers about 40,000 base pairs, i.e., 1% of the Salmonella chromosome, with each transduction event. Pepending on the recombination event that ensues, small amounts, or even all, of this introduced DNA will become part of the chromosome of the S. typhi recipient. This could lead to many silent changes to the S. typhi chromosome that adversely affect vaccine potential.
  • the silent transposon insertion zib-908::Tn10 has also been used to introduce delpurA155 into S. typhi (U.S. Patent 4,735,801; and Edwards et al, J. Bacteriol., 170:3991-3995 (1989)). Selection for tetracycline-sensitive derivatives using fusaric acid-resistance, removed the Tn10 insertion. Unfortunately, spontaneous excision of Tn10 in the majority of cases can lead to inversion and/or deletion of adjacent DNA (Ross et al, Cell, 16:721-731 (1979); and Kleckner, In: Mobile DNA, Berg et al, eds., American Society for Microbiology, Washington, D.C., pp. 227-268 (1989)). Thus, strains made in this fashion might carry an undefined inversion and/or deletion at the point where Tn10 excision occurred.
  • Chorismic acid is an important biochemical precursor of the aromatic amino acids tryptophan, tyrosine and phenylalanine, ubiquinone and menaquinone, and the vitamins p-aminobenzoic acid (hereinafter “PABA”) and 2,3-dihydroxybenzoic acid (hereinafter “DHB”) (Pittard, In: Escherichia coli and Salmonella typhimurium, Neidhardt, ed., American Society for Microbiology, Washington, D.C. (1984)).
  • PABA p-aminobenzoic acid
  • DDB 2,3-dihydroxybenzoic acid
  • Ubiquinone and menaquinone are required for H 2 S production and oxidative phosphorylation (Poole et al, In: Escherichia coli and Salmonella typhimurium, Neidhardt, ed., American Society for Microbiology, Washington, D.C. (1984)).
  • PABA is required for the synthesis of folate which is then used for the synthesis of glycine from serine, methionine, formyl-met-tRNA, and thymine
  • enterochelin an iron chelator produced by Salmonella
  • enterochelin an iron chelator produced by Salmonella
  • Salmonella Stocker et al, Abstr. Ann. Mtg. Amer. Soc. Microbiol. (1979); and Stocker et al, Develop. Biol. Stand., 53:47-54 (1983)
  • enterochelin is a virulence factor of S. typhi (Stocker et al, Curr. Top. Microbiol. Immunol., 124:149-172 (1986)).
  • S. typhimurium aroA mutants are incapable of chorismate synthesis and are attenuated when given to mice either orally or intraperitoneally (Stocker et al, Abstr. Ann. Mtg. Amer. Soc. Microbiol. (1979); Hoiseth et al, Nature, 291:238-239 (1981); U.S. Patent 4,837,151; Smith et al, Am. J. Vet. Res., 45:1858-1861 (1984); and O'Callaghan et al, Infect. Immun., 56:419-423 (1988)).
  • aroF, aroG and aroH genes which encode isoenzymic forms of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which catalyze the first step of chorismate synthesis, i.e., conversion of phosphoenol pyruvate and erythrose 4-phosphate to 3-deoxy-D-arabino- heptulosonate 7-phosphate; and the aroL gene and an unidentified gene that encode Shikimate kinase II and Shikimate kinase I, respectively, which catalyze the fifth step of chorismate synthesis, i.e., conversion of shikimate to shikimate 3-phosphate (Pittard, In: Escherichia coli and Salmonella typhimurium, Neidhardt, e
  • a method for the construction of strains of Salmonella harboring two independent aro mutations in different aro genes, in particular, construction of aroA and aroC double mutants of Salmonella (Pougan et al, Mol. Gen. Genet., 207:403-405 (1987); and Pougan et al, J. Infect. Dis., 158:1329-1335 (1988)).
  • the method involved the introduction of a the first deletion using P22-mediated transduction of aroA::Tn10 into the target Salmonella strain and selection for the loss of Tn10 in the aro mutant by imprecise excision of the transposon resulting in tetracycline-sensitivity and aromatic auxotrophy.
  • kanamycin-resistance was used as positive selection for the introduction of the second mutant allele.
  • Mercury-resistance could be used in place of kanamycin-resistance for the development of human vaccine, since kanamycin-resistance is an unacceptable property for a live oral typhoid fever vaccine (Pougan et al, J. Infect. Dis., 158:1329-1335 (1988)).
  • European Patent Publication 322237 describes a similar method for the construction of double aro mutants. The method thereof relies on the use of imprecise excision for the removal of antibiotic-resistance markers during the construction of the mutants.
  • Specific examples of double aro mutants described therein are S. typhi aroA aroE and S. typhi aroA aroC double mutants.
  • the attack rate of clinical illness that follows ingestion of bacterial enteropathogens and the severity of clinical illness is a function of the inoculum size, i.e., the number of viable organisms ingested (Hornick et al, N. Engl. J. Med., 283:686-691 (1970); and Levine et al, In: Acute Enteric Infections in Children: New Prospects for Treatment and Prevention, Holme et al, eds., Amsterdam, Elsevier/North Holland, pp. 443-459 (1981)).
  • the critical factor is the number of pathogenic bacteria that survive transit through the gastric acid barrier to reach the small intestine. That is, when fasting volunteers ingested 10 6 V.
  • the gastric acid barrier that represents a potent non-specific defense against bacterial enteropathogens also plays an important role in determining the immunogenicity and protective efficacy of live oral enteric vaccines.
  • Shigella live oral vaccines are highly efficacious when children ingested the vaccine with bicarbonate buffer to ensure survival through the gastric acid barrier, but are not protective when the vaccine is given without buffer (Mel el al, Bull. Wld. Hlth. Org., 32:647-655 (1965); and Mel et al, Bull. Wld. Hlth. Org., 45:457-464 (1971)).
  • Galenic formation plays a critical role of the vaccine in determining immunogenicity and efficacy.
  • Pifferent formulations relate to strategies by which vaccine organisms, following ingestion, are transported through the stomach to reach the small intestine in a viable state.
  • Ty21a is an attenuated strain of S . typhi (Germanier et al, J. Infect. Dis.,
  • the increased protection conferred by the four doses cannot be due to a booster effect but, rather, must be due to delivering more viable attenuated bacteria to the small intestine from whence, upon release of the capsules, the bacteria can translocate to the lamina intestinal and mesenteric lymph nodes as a prerequisite to further spread within the reticuloendothelial system.
  • the requirement for multiple doses of Ty21a is reflected in the discovery in the present invention of the sensitivity of Ty21a to low pH. Thus, to overcome this sensitivity, large numbers of organisms must be given in multiple doses to achieve an effective immune response.
  • the macrophage is a professional phagocytic cell that takes up antigens, including microorganisms, for their removal from the host (Metchnikoff, In: Lectures in Comparative Pathology of Inflammation. Kegan, Paul, Trench, Truber and Co., London (1893); and Moulder, Microbiol. Rev., 49:298-337 (1985)) .
  • the phagocytic process involves the binding of antigen to receptors on the macrophage surface, either to the C3 receptor or to the Fc receptor, which causes the membrane to invaginate and engulf the antigen into what is called the phagosome.
  • the phagosome is internalized into the cytoplasm of the macrophage where, under normal circumstances, it becomes fused to the lysosomes. After fusion, the lysozymes release their antigen-inactivating contents into the phagosome (Klebanoff, In: Inflation: Basic Principals and Clinical Correlates. Gallin et al, eds., Raven, New York (1988); and Elsbach et al, In: Inflation: Basic Principals and Clinical Correlates. Gallin et al, eds., Raven, New York (1988)).
  • S . typhi are representative of a group of bacteria that survive for extended periods within macrophages and elicit cell-mediated immunity (Olitzki et al. Path. Microbiol. (Basel), 27:175-201 (1964); and Levine et al, J. Clin. Invest., 79:888-902 (1987)).
  • Other bacteria of this type include non-typhoidal Salmonella spp, Mycobacteria spp, Listeria monocytogenes, Francisella spp, Legionella spp and Rickettsia spp (Moulder, Microbiol. Rev., 49:298-337 (1985)).
  • typhi does not employ either of these two methods, but instead is able to survive the killing effect of lysosome-derived cationic peptides, called defensins, H 2 O 2 and oxygen radicals (also released by the lysosome into the phagosome), and acidification of the phagosome by a yet to be elucidated mechanism (Olitzki et al. Path. Microbiol. (Basel), 27:175-201 (1964)).
  • the mutations mapped in the phoP gene The phoP gene, which along with the phoQ gene, has been shown to be a regulator of several genes, including transcriptional activation of the pagA, pagB and pagC genes, and repression of another unidentified group of genes, called the prg genes (Miller et al, J.
  • phoP/phoQ activated genes are thought to provide S. typhimurium with resistance to macrophage-derived defensins (Groisman et al, Proc. Natl. Acad. Sci. USA, 86:7077-7081 (1989); Miller et al, Proc. Natl. Acad. Sci. USA, 86:5054-5058 (1989)).
  • the fact that phoP and phoQ mutants were also found to be attenuated reflects the importance of the resistance to macrophage killing in the pathogenesis of S. typhimurium (Miller et al, Proc. Natl. Acad. Sci. USA, 86:5054-5058 (1989)).
  • Bacteriol., 130:420-428 (1977)) is introduced into a mouse-virulent strain of S. typhimurium by P22-mediated transduction (Miller et al, J. Bacteriol., 172:2485-2490 (1990)), the resulting strain becomes as attenuated as phoP/phoQ mutants, but remains resistant to defensins (Miller et al, J. Bacteriol., 172:2485-2490 (1990)).
  • the phoP c strain was also found to be highly immunogenic when given intraperitoneally, even at doses as low as 15 viable organisms. However, the phoP c mutation remains genetically undefined, other than it maps at the phoP/phoQ locus.
  • Resistance to oxidative stress can be induced by exposure of S. typhimurium to low levels of H 2 O 2 .
  • This response is under the regulation of oxyR (Christman et al, Cell, 41:753-762 (1985)).
  • An adaptative response like this one might provide S. typhimurium with an additional mechanism to escape the killing caused by the oxidative burst within the macrophage.
  • Several proteins that are induced as a consequence of the H 2 O 2 adaptative response have been identified.
  • S . typhimurium Another adaptative response that is well described in S . typhimurium is the heat shock response (Neidhardt et al, In: Escherichia coli and Salmonella typhimurium, Neidhardt, ed., American Society for Microbiology, Washington, D.C. (1984)).
  • heat shock stress e.g., growth at 37°C to 42oC
  • the regulation of many of the cell's protein and carbohydrate synthesis pathways becomes altered.
  • a class of proteins called the heat shock proteins are induced during such a response.
  • Mycobacterium leprae and Mycobacterium tuberculosis also have been shown to express heat shock response proteins, particularly a 65kP protein which is homologous to the E. coli heat shock protein, GroEL, and is the immunodominant antigen of these two pathogens.
  • the expression of the 65kP GroEL-like protein of M. leprae and M. tuberculosis in the macrophage is thought to induce a specific cell-mediated immune response and lead to macrophage lysis (Shinnick et al, Infect. Immun., 56:446-451 (1988)).
  • Such a process might provide M. spp with a means to spread to neighboring cells (Kaufmann, Immunol Today, 9:168-173 (1988)).
  • Listeriolysin a major virulence factor of Listeria monocytogenes, is under the control of the heat shock protein response and is expressed within the macrophage (Sokolovic el al, Infect. Immun., 57:295-298 (1989)). Since S. typhimurium produces the same heat shock response proteins, and in addition, the groEL proteins play a role in survival within the macrophage (Buchmeier, Abstr. Ann. Mtg. Amer. Soc. Microbiol. (1990)), it is possible that other heat shock proteins are involved in virulence.
  • ATR acid-tolerance response
  • S. typhimurium causes gastroenteritis and the growth of the bacilli in humans is restricted mainly to the intestinal lumen and mucosal layer.
  • S . typhimurium invades the mucosa and is phagocytosed by polymorphonuclear cells that infiltrate the site of invasion, not by the macrophages, as is the case for S.
  • S. typhimurium harbors large plasmids (typically about 90,000 base pairs, which represents 2% extra genetic material), called the virulence plasmid because of its virulence enhancing properties.
  • Strains of S. typhimurium that have lost the virulence plasmid are attenuated in mice (Gulig et al, Infect. Immun., 55:2891-2901 (1987)). There are at least two regions that express this virulence enhancing property (Norel et al, Molec. Microbiol., 3:733-743 (1989)).
  • the plasmid has not been found to be associated with the ability of
  • an object of the present invention is to provide acid-tolerant S. typhi.
  • a further object of the present invention is to provide double aro mutants of S. typhi.
  • Another object of the present invention is to provide an oral vaccine against typhoid fever.
  • Still another object of the present invention is to provide a single dose oral vaccine against typhoid fever.
  • Yet another object of the present invention is to provide a single dose oral vaccine against typhoid fever which is useful as a carrier of genes expressing foreign antigens cloned from other pathogens and that raises protective immune responses in humans against the pathogen from which the foreign antigens were derived.
  • An additional object of the present invention provide a method for immunizing a subject against typhoid fever.
  • a further object of the present invention is to provide a method for simultaneously immunizing a subject against typhoid fever and another pathogen.
  • Another object of the present invention is to provide a method for enrichment of acid-tolerant S. typhi.
  • Yet another object of the present invention is to provide a method for enrichment of acid-tolerant attenuated S. typhi useful as a live oral typhoid fever vaccine.
  • An additional object of the present invention provide a method for enrichment of acid-tolerant attenuated S. typhi expressing cloned foreign antigens useful as a carrier vaccine.
  • a vaccine against typhoid fever comprising:
  • the above-described objects of the present invention have been met by a method of immunizing a subject against typhoid fever comprising orally administering a pharmaceutically effective amount of an acid-tolerant typhoid fever vaccine strain of Salmonella typhi.
  • the above-described objects of the present invention have been met by a method of immunizing a subject against typhoid fever comprising orally administering a pharmaceutically effective amount of an aroC, aroD double mutant of Salmonella typhi.
  • Salmonella typhi comprising the steps of:
  • step (ii) determining viability of the cultured Salmonella typhi of step (i), wherein if the cultured Salmonella typhi of step (i) is viable;
  • step (C) harvesting the resulting Salmonella typhi of step (A), so as to isolate and enrich for acid-tolerant Salmonella typhi.
  • Figure 1 shows a Bglll-linearized scaled diagrammatic representation of plasmid pCVD1001 (8.2 kb) and its restriction map.
  • Plasmid pCVD1001 was derived from pAROD-TYPHI, a 17.6 kb cosmid constructed in the 6.4 kb cosmid vector pHC79. The position and relative size of the 0.756 kb coding region of the aroD gene is also shown. Further, the position of the transposon Tn1725 (9.0 kb) insertion in pCVD1001 that resulted in plasmids pCVD1006 (17.2 kb) and pCVP1007 (17.2 kb) are shown.
  • Transposon Tn1725 has two EcoRI digestion sites, each located 15 bp from the transposon ends. Therefore, a Tn1725 insertion effectively creates a EcoRI digestion site.
  • Figure 2 shows a BamHI- and Bglll-linearized scaled diagrammatic representation of plasmids pCVD1010 (5.35 kb) and pCVD1013 (8.45 kb) and their restriction maps.
  • Plasmid pCVD1010 carries the 0.75 kb Hindlll (converted to SaIl) to EcoRI (located in the Tn1725 insertion) fragment of pCVD1006 and the 0.9 kb EcoRI (located in the Tn1725 insertion) to BamHI fragment of pCVD1007. These fragments were joined by ligation at the EcoRI site.
  • the resulting fused fragment form a 1.65 kb hybrid fragment which is flanked by SaIl and BamHI sites, and is inserted into SaIl- and Bglll-linearized pGP704 (3.7 kb).
  • the BamHI and BgllI sites are lost as a result and the fusion point is designated as "F”.
  • Plasmid pCVD1013 is identical to pCVD1010, but the 3.8 kb Pstl fragment of pKTN701 (6.8 kb), which carries the cat gene, has replaced the 0.7 kb Pstl fragment of pCVP1010, which carries the bla gene of pGP704.
  • Figure 3 shows an EcoRI-linearized scaled diagrammatic representation of pCVD1003 (6.4 kb) and its restriction map.
  • the fusion joint of the BallI ends of pBRD138 and the BamHI site of pUC9 is designated as "F”.
  • the location of the 1.077 kb aroC gene of S. typhi. which spans from 124 bp upstream of the Pstl site in the S. typhi PNA to 299 bp downstream of the Nrul site, also located in the S. typhi DNA, is shown.
  • Plasmid pCVD1015 (not shown) has the SaIl fragment of pCVD1003 deleted. Plasmid pCVD1015, which has a unique Nrul site, was further derivised by deleting the 0.654 kb Pstl to Nrul fragment located in the aroC gene to create pCVD1016 (not shown). The 0.654 kb Pstl to Nrul deletion in plasmid pCVD1016 results in an altered aroC gene, designated delaroC1019.
  • Figure 4 shows an EcoRI-linearized scaled diagrammatic representation of plasmids pCVP1011 (5.3 kb) and pCV1019 (6.7 kb) and their restriction maps.
  • Plasmid pCVD1011 was derived by inserting the 1.6 kb SaIl to EcoRI fragment of pCVD1016 (which carries delaroC1019) into Sail- and EcoRI-linearized pGP704.
  • the deletion modified aroC gene, designated delaroC1019. located on pCVD1019, is shown as " ⁇ C".
  • Plasmid pCVD1019 is identical to pCVD1011, but with the 4.6 kb BamHI to Pstl fragment, which carries the cat gene of pKTN701 has replaced the 2.1 kb Pstl fragment of pCVD1011, which carries the bla gene of PGP704.
  • Figure 5 shows the series of events that led to the introduction of delaroC1019 into the chromosome of S . typhi.
  • plasmid pCVD1019 is introduced into S. typhi by electroporation, whereupon it undergoes a homologous recombination event.
  • plasmid pCVD1019 becomes integrated into the chromosome of S. typhi. This results in formation of a stable cointegrate (H236.1) This cointegrate is resistant to chloramphenicol .
  • a second homologous recombination event results in the curing of the cointegrate. There are two possible outcomes of this second recombination event.
  • the first (1.) is due to recombination on the same side of delaroC1019 as the first recombination event. This results in a aroC + genotype (H239.1).
  • the second (2.) is the result of recombination on the opposite side of delaroC1019 as the first recombination event. This results in a delaroC1019 genotype (H238.1). Plasmid pCVD1019 cannot replicate in S. typhi and thus is lost after replication.
  • Figure 6 shows the nucleotide sequence of the aroC gene of S. typhi.
  • the coding region for the aroC gene starts with an ATG codon (position 1) and ends with a TGG codon (position 1077).
  • the deletion mutation delaroC1019 is underlined and is flanked by a Pstl site (CTGCA 124 G; digests at position 124) and an
  • Nrul site TCG 778 CGA; digests at position 778.
  • Figure 7 shows the nucleotide sequence of the aroD gene of S. typhi.
  • the coding region of the aroD gene starts with an ATG codon (position 1) and ends with a GCC codon (position 775).
  • # denotes that the preceding underlined sequence gives the approximate position of the Tn1725 insertion in pCVD1007.
  • * denotes that the preceding underlined sequence gives the approximate position of the Tn1725 insertion in pCVD1006.
  • the position of the EcoRV digestion site is shown for reference to Figure 1.
  • the above-described objects of the present invention have been met by substantially pure acid-tolerant Salmonella typhi.
  • a vaccine against typhoid fever comprising:
  • a vaccine against typhoid fever comprising: (A) a pharmaceutically effective amount of an aroC, aroD double mutant of Salmonella typhi; and
  • the above-described objects of the present invention have been met by a method of immunizing a subject against typhoid fever comprising orally administering a pharmaceutically effective amount of an acid-tolerant typhoid fever vaccine strain of Salmonella typhi.
  • the above-described objects of the present invention have been met by a method of immunizing a subject against typhoid fever comprising orally administering a pharmaceutically effective amount of an aroC, aroD double mutant of Salmonella typhi.
  • Salmonella typhi by:
  • step (i) culturing an aliquot of the resulting Salmonella typhi of step (A) at a selective pH of about 2.0 to 3.5 at about 30 to 40oC;
  • step (ii) determining viability of the cultured Salmonella typhi of step (i), wherein if the cultured Salmonella typhi of step (i) is viable; (C) harvesting the resulting Salmonella typhi of step (A), so as to isolate and enrich for acid-tolerant Salmonella typhi.
  • a method for the enrichment of acid-tolerant S. typhi has been designed so as to obtain "substantially pure" acid-tolerant S . typhi.
  • substantially pure acid-tolerant S. tyhpi means that greater than about 75% of the organisms survive after 90 min at pH 3.0.
  • Enrichment of acid-tolerance of S . typhi is believed in the present invention to increase survival of S. typhi and its attenuated derivatives during their passage through the gastric environment of the stomach and within the acidified phagolysosome of infected macrophages, thereby enabling more viable organisms to reach the site of invasion and enhancing the ability of the vaccine strain to stimulate cell-mediated immunity.
  • the acid-tolerant S . typhi of the present invention can be used to produce a live oral typhoid fever vaccine.
  • any mutation or any combination of known mutations that cause safe attenuation, but retain effective immunogenicity can be introduced into acid-tolerant wild-type S. typhi.
  • the particular wild-type S. typhi strain from which the acid-tolerant vaccine is derived and which can be employed in the immunization method of the present invention is not critical thereto.
  • the wild-type S. typhi may be any well-known recently isolated strain, such as the ISP1804, ISP1820, ISP2822, ISP2825, Chilean and Thai strains (Murray et al, J. Infect. Dis., 151:551-555 (1985) and Peruvian strains (Gotuzzo et al, J. Clin. Microbiol., 25:1779-1781 (1987)).
  • Mutations that cause safe attenuation, but retain effective immunogenicity can be introduced using non-specific mutagenesis, such as N-methyl-N'-nitro-N- nitrosoguanidine; classic genetic techniques such as Tn10 mutagenesis, P22-mediated transduction 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 mutations that cause safe attenuation, but retain effective immunogenicity include:
  • auxotrophic mutations such as aro (Hoiseth et al, Nature, 291:238-239 (1981)), gua (McFarland et al, Microbiol. Path.,
  • Preferable mutations are those that result in safe attenuation but do not affect immunogenicity.
  • Pouble aroC, aroD mutants are most preferred.
  • the double mutants may be insertion mutants, deletion mutants or a combination thereof, although deletion mutants are preferred.
  • the aroC gene is 1.077 kb in size (see Figure 6).
  • the size of the deletion in the aroC mutant may range from 0.001 to 100 kb, preferably, from 0.001 to 1.077 kb.
  • intracistronic deletions in aroC can range from 1 base pair to 1.077 kb in size, deletions can also be made that extend beyond the aroC gene, i.e., extracistronic deletions of up to 100 kb. However, the latter is not preferable.
  • the aroD gene is 0.755 kb in size (see Figure 7).
  • the size of the deletion in the aroD mutant may range from 0.001 to 100 kb, preferably, from 0.001 to 0.755 kb.
  • intracistronic deletions in aroD can range from 1 base pair to 755 base pairs in size, deletions can also be made that extend beyond the aroD gene, i.e., extracistronic deletions of up to 100 kb. However, the latter is not preferable.
  • Peletions can be made in the S. typhi aroC gene using convenient restriction sites, such as Asul (position 121), Bbvl (positions 311, 955), BssHII (positions 753, 755, 757), Smal (position 487), and Xmal (position 485) or by site-directed mutagenesis with oligonucleotides (Sambrook et al, eds., In: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Publications (1989)).
  • Peletions can be made in the S. typhi aroD gene using convenient restriction sites, such as Alul (positions 118, 449), Clal (positions 337, 785), EcoRV (position 161), Fokl (position 53) and Pdel (position 180) or by site-directed mutagenesis using oligonucleotides (Sambrook et al, eds., In: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Publications (1989)).
  • Inactivation of the aroC gene and aroD gene can also be carried out by an insertion of foreign DNA using any of the above-mentioned restriction sites or by site-directed mutagenesis with oligonucleotides
  • the insertion can be made anywhere inside the aroC or aroD gene coding region or between the coding region and the promoter.
  • aroD or aroC inactivation include transfer into S . typhi deletions or insertions made in S. typhimurium aroC or aroD genes or E. coli aroC or aroD genes, transposon-generated deletions, and imprecise excision of DNA insertions. The latter two methods are more likely to make deletions that extend beyond the aroD or aroC gene and therefore are not preferable,
  • acid-tolerance can be introduced into a live oral typhoid fever vaccine strain by transfer of DNA sequences encoding acid-tolerance.
  • genetic basis of acid-tolerance has not yet been determined, those skilled in the art can readily identify the gene or genes involved using conventional techniques, such as transposon mutagenesis, transcriptional fusions in addition to gene cloning and sequencing methodologies.
  • the cloned genes can then be introduced into a vaccine strain of S .typhi, or a wild-type strain of S . typhi which is employed to produce a vaccine strain, by recombinational crosses or as part of an expression cassette on plasmid vectors.
  • the particular S. typhi vaccine strain from which the acid-tolerant S. typhi vaccine strain is derived and which can be employed in the immunization method of the present invention is not critical thereto.
  • the S . typhi vaccine strain may be any well-known vaccine strain, such as the Ty21a, Ty21a-Vi + , 541Ty, 543Ty, 27V strains, in addition to the CVD906 strain developed in the present invention.
  • Another alternative is to identify the gene regulator of acid-tolerance using transposon mutagenesis and other genetic methodologies, such as gene transfers between S. typhimurium and S . typhi, and to alter the regulator in such a way as to cause constitutive expression of acid-tolerance in the resulting S. typhi strain. If the regulator controls gene expression by a negative control, then deletion inactivation would result in constitutive expression of acid-tolerance. If the regulator controls gene expression by a positive control, then those skilled in the art can design modifications that cause an irreversible gene activation by the repressor that would result in constitutive expression of acid-tolerance.
  • the S. typhi of the present invention can also be used as carriers of genes expressing protective antigens cloned from other pathogens.
  • protective antigens means antigens or epitopes thereof which give rise to protective immunity against infection by the pathogen from which they are derived.
  • pathogens from which genes encoding protective antigens would be cloned are not critical to the present invention.
  • examples of such other pathogens include protozoan, viral and bacterial pathogens.
  • protective antigens of protozoan 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. falciparum; and gp63 of Leishmania spp. (Russell et al, J. Immunol., 140:1274-1278 (1988)).
  • protective antigens of viral pathogens include the hepatitis B surface antigen (Wu et al, Proc. Natl. Acad. Sci. USA. 86:4726-4730 (1989)); human immunodeficiency virus antigens, such as gp120, gp41 (Ratner et al. Nature, 313:277-280 (1985)) and T cell and B cell epitopes of gp120 (Palker et al, J. Immunol., 142:3612-3619 (1989)); and 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)).
  • VP4 Mackow et al, Proc. Natl. Acad. Sci. USA, 87:518-522 (1990)
  • 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 (hereinafter "ETEC"), such as the CFA/I fimbrial antigen (Yamamoto et al, Infect.
  • ETEC enterotoxigenic E. coli
  • Antigens introduced into the carrier must be well-expressed. This can be achieved by constructing an expression cassette which includes a transcriptional promoter, a ribosomal binding site, and a start codon fused to the gene of interest.
  • promoters useful in the present invention include the lambda P L , ptac. ptrp or plac promoters (Hoopes et al, In: Escherichia coli and Salmonella typhimurium, Neidhardt, ed., American Society for Microbiology, Washington, D.C. (1984); Reznikoff et al, In: Maximizing Gene Expression. Reznikoff, ed., Butterworths (1986); and Miller et al, eds., In: The Operon, Cold Spring Harbor Publications (1978)).
  • Stability of the antigen in the carrier can be accomplished by the well-known technique of chromosomal integration (Hone et al, Microbial. Path., 5:407-418 (1989)) or the use of plasmid complementation systems (Nakayama et al, Bio/Tech, 6:693-697 (1988)).
  • S. typhi of the present invention to be administered will vary depending on the age, weight and sex of the subject. Generally, the dosage employed will be about
  • 5.0 x 10 3 to 5.0 x 10 11 viable organisms preferably about 1.0 x 10 5 to 5.0 x 10 9 viable organisms.
  • the particular pharmaceutically acceptable carrier or diluent employed is not critical to the present invention.
  • diluents include 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. II:467-470 (1988)).
  • carriers include proteins, e.g., as found in skim milk, sugars, e.g. sucrose, or polyvinylpyrrolidone.
  • the method of the present invention depends on the ability of S. typhi strains to initially adapt to acidic conditions, i.e., growth at about pH 4.5 to 5.5, preferably about pH 4.9 to 5.1, more preferably about pH 5.0, without aeration, i.e., in an oxygen tension of less than about 1.0% (v/v) to anaerobic conditions, at about 20 to 42oC, preferably about 35 to 39oC, generally to an OD 600 of about 0.2 to 0.5, preferably about 0.3.
  • the method described is one which gives the greatest enrichment of acid-tolerance.
  • the pH range is important since the peak of acid-tolerance enrichment occurs at pH 5.0 ⁇ 0.1.
  • the oxygen tension is important since only low acid-tolerance enrichment occurs when S. typhi is grown in the presence of greater than 1.0% (v/v) to atmospheric pressure oxygen.
  • the temperature that best facilitates acid-tolerance enrichment is 37oC ⁇ 2oC. However, other temperatures, while not optimal, do allow some enrichment of acid-tolerant S . typhi.
  • the OD 600 ranges give highest acid-tolerance enrichment. However, enrichment does occur at other OD 600 values.
  • the bacteria are grown at a selective pH, about the acidic levels of the stomach at the time of immunization, i.e., at about pH 2.0 to 3.5, preferably about pH 2.9 to 3.1, at about 30 to 40oC, preferably about 35 to 39°C, and viability is determined, e.g., as described in In: Manual of Methods for General Bacteriology. American Society for Microbiology, Gerhardt et al, eds., Washington, D.C. (1981).
  • the S. typhi can be cultured in any well-known nutrient medium, such as L-broth (Pifco, Petroit, MI), Brain-Heart Infusion (Pifco, Petroit, MI), Nutrient broth (Pifco, Detroit, MI), M9 medium (Miller, In: Experiments in Molecular Biology, Cold Spring Harbor Press (1979)) supplemented with 0.2% (w/v) casamino acids (Difco, Petroit, MI), Peptone broth (Difco, Petroit, MI) and Tryptone broth (Pifco, Petroit, MI).
  • L-broth Pifco, Petroit, MI
  • Brain-Heart Infusion Pifco, Petroit, MI
  • Nutrient broth Pifco, Detroit, MI
  • M9 medium M9 medium (Miller, In: Experiments in Molecular Biology, Cold Spring Harbor Press (1979) supplemented with 0.2% (w/v) casamino acids (Difco, Petroit, MI), Peptone broth (Difco, Petroit,
  • the S. typhi of the present invention are stored at -70oC suspended in 50% (v/v) glycerol, 0.5% (w/v) Tryptone, 0.25% (w/v) Yeast extract, 0.01% (w/v) PABA, and 0.1% (w/v) casamino acids (pH 5.0), or lyophilized in 5.0% (w/v) sucrose in Aro-Broth (pH 5.0).
  • S. typhimurium. strain LT2 can be obtained when these bacilli are grown with aeration in minimal E medium comprising 0.2% (w/v) MgSO 4 .7H 2 O, 0.2% (w/v) citric acid.1H 2 O, 1.0% (w/v) K 2 HPO 2 anhydrous, and 0.35% (w/v) NaHNH 4 PO 4 .4H 2 O (pH 7.6) as compared to when grown in minimal E medium (pH 5.8) (Foster et al, J. Bacteriol., 172:771-778 (1990)).
  • minimal E medium comprising 0.2% (w/v) MgSO 4 .7H 2 O, 0.2% (w/v) citric acid.1H 2 O, 1.0% (w/v) K 2 HPO 2 anhydrous, and 0.35% (w/v) NaHNH 4 PO 4 .4H 2 O (pH 7.6) as compared to when grown in minimal E medium (pH 5.8) (Foster
  • Viability counts were performed on duplicates of each sample. The viability count is the number of colonies on a given plate multiplied by 10 (to convert the results to counts per ml of sample) and multiplied by the dilution factor (to convert the results to the original sample numbers). Viability counts were taken at 0 min and 90 min after the cells were suspended in the pH 3.0 medium. The results were expressed as the number of viable organisms at 90 min divided by the number of viable organisms at 0 min and multiplied by 100 so as to express the result as a % survival. The results are shown in Table 1 below.
  • CFB medium comprising 0.15% (w/v) Yeast extract (Pifco, Petroit, MI), 1.0% (w/v) casamino acids (Pifco, Petroit, MI), 0.005% (w/v) MgSO 4 (Sigma, St. Louis, MO) and 0.005% (w/v) MnCl 2 (Sigma, St. Louis, MO), at 25oC has been shown to enhance expression of V. cholerae virulence factors (Hall et al, Microbial. Path., 4:257-265 (1988)).
  • the capacity of CFB medium to produce cultures highly enriched with acid-tolerant S. typhi was tested as follows.
  • L-broth the pH of which was adjusted to pH 7.2 using 10 M NaOH and to pH 5.0 using 10 M HCl, is a standard laboratory medium used for growth of Salmonella and other enteric bacteria. Thus, L-broth was tested for its capacity to produce cultures highly enriched for acid-tolerant S. typhi. The method employed was identical to that described above for growth in CFB medium. The results are shown in Table 3 below.
  • H28 Oletyphi isolated from different geographic regions, H28 (Originated from India), H29 (Originated from Malaysia) and H30 (Originated from the Solomon Islands), and previous vaccine strains, Ty21a and 541Ty, proved to be inherently more acid-sensitive.
  • Strains H28, H29 and H30 were provided by J. Morris, Pepartment of Microbiology, University of Melbourne (1986).
  • the results above indicate that maximum yields of acid-tolerant S. typhi is dependent on two key factors.
  • Second, the strain is preferably a recent isolate that is capable of becoming acid-tolerant after growth in the appropriate medium.
  • the cultures grown without aeration were harvested after 20 hr of incubation. At this time, the OD 600 was typically about 0.5.
  • the cells were pelleted, washed and resuspended in L-broth (pH 3.0) as described for the aerated cultures. Immediately after the cells were resuspended in L-broth (pH 3.0), a 0.1 ml sample was taken and transferred to 0.9 ml of PBS.
  • the pH 3.0 cell suspensions were placed at 37°C, standing with 1.0 ml of mineral oil overlaying the culture or in an anaerobic atmosphere by placing the cultures in a GasPak ® Jar System (BBL, Cockeysville, MD) with an anaerobic GasPak ® (BBL, Cockeysville, MD). Then, after 90 min, the 1.0 ml samples were taken and mixed with 1.0 ml of PBS. Viability counts were determined for each of the 0 min and 90 min samples as described above. The results are shown in Table 5 below.
  • the yield of acid-tolerant S . typhi is increased by dropping the pH to 5.0.
  • S. typhi is grown without aeration the yield of acid-tolerant S. typhi is maximized.
  • S. typhi. strain ISP1820 is grown at 37oC in L-broth (pH 5.0) without aeration to an OD 600 of 0.5, 125% of the bacilli are able to survive L-broth (pH 3.0) for 90 min.
  • Strain CVP906 which is a double aro mutant of ISP1820 and was prepared as described in more detail below, gave rise to a higher yield of acid-tolerance when using the maximized conditions described for the parent strain, ISP1820. However, CVP906 is less acid-tolerant than its parent. This difference might the result of the laboratory passage CVP906 underwent during its construction.
  • Attenuated candidate vaccine strains of S. typhi have been prepared using classical genetic techniques (Edwards et al, J. Bacteriol., 170:3991-3995 (1988)).
  • the strains described herein were made using recombinant DNA techniques.
  • typhi strain ISP1820 to produce strain CVP906 are delaroC1019 and delaroD1013. These aro deletions are defined in molecular terms, they do not extend beyond the limits of the mutant gene and no other foreign DNA (except 30 base pairs of innocuous DNA from transposon Tn1725) was introduced.
  • Attenuated strains of S. typhi for use as a typhoid fever vaccine and antigen carrier, preferably possess two well-separated and well-defined deletion mutations and preferably are highly immunogenic after a single dose.
  • strains of Salmonella harboring a single aro mutation are remarkably attenuated but highly immunogenic in animals.
  • S . typhi strains that carry both aro and pur mutations were shown to be safe but non-immunogenic in volunteers and hence hyperattenuated.
  • the pur mutation included as a safety factor, has been shown to cause the hyperattenuation in mice given pur and aro, pur strains of S. typhimurium.
  • aro mutations on the Salmonella chromosome are widely separated, mutations in two of the aro genes can be exploited to introduce safety against the chance of in vivo reversion, as was attempted with the aro pur double mutants of S. typhi. It is proposed herein to use aro mutations as the primary attenuating marker of S . typhi with the aim of constructing a live oral typhoid fever vaccine that is also suitable as an antigen carrier. To this end, in vitro DNA techniques were used to construct defined deletions in the aroC and aroD genes of acid-tolerant S . typhi strain ISP1820.
  • Electrophoresis of DNA was carried out on 0.5-1.0% (w/v) agarose gels buffered with TAE buffer comprising 0.04 M
  • the molecular weight markers employed were the "1.0 kb ladder” (Bethesda Research Laboratories, Gaithersburg, MD).
  • the molecular weight of restriction enzyme generated fragments was determined from a standard curve of the log 10 molecular weight of the 1.0 kb ladder standard sized fragments versus their mobility in the agarose gel. After electrophoresis, the isolation of restriction enzyme-generated fragments from agarose was achieved using the Gene Clean Kit ® (Bio-101, Richmond, CA) as recommended by the manufacturer.
  • Plasmid pCVD1001 (see Figure 1) was derived from pAROD-TYPHI (G. Pougan, Wellcome Biotech, Beckenham Kent, England), a 17.6 kb cosmid constructed in the 6.4 kb cosmid vector pHC79. Plasmid pCVD1001 was constructed by digesting pAROD-TYPHI with Bglll, which produced 4 fragments sized 8.2, 5.7, 2.1 and 1.6. The largest fragment (8.2 kb) carries sufficient pHC79 PNA (2.7 kb) to enable plasmid replication and expression of ampicillin-resistance, in addition, to 5.5 kb of S. typhi DNA which carries the aroD gene. Thus, digestion of pAROD-TYPHI with Bglll followed by recircularization of the 8.2 kb fragment by ligation results in a new plasmid, designated pCVP1001.
  • Transposon Tn1725 was used to map the location of the aroD gene in pCVD1001 relative to restriction endonuclease sites located in pCVD1001 using the method described by Ubben et al. Gene, 41:145-152 (1986).
  • Plasmids pCVD1006 and pCVD1007 were used to construct an altered aroD gene (see Figure 2).
  • Transposon Tn1725 has EcoRI digestion sites located 15 base pairs from each end. Thus, an insertion using this transposon effectively creates an EcoRI site that can be utilized to create new enzyme-derived fragments (Ubben et al. Gene, 41:145-152 (1986)).
  • the 0.9 kb EcoRI to BamHI fragment of pCVD1007 spanning from the BamHI site within pCVD1007 and upstream of the aroD gene, to the EcoRI site within Tn1725 (see Figure 1), was isolated and purified. More specifically, pCVD1007 was digested with BamHI and EcoRI simultaneously and the resulting DNA fragment was electrophoresed without further modification. The 0.9 kb EcoRI to BamHI fragment was excised from the agarose gel after electrophoresis and purified.
  • the resulting ampicillin-resistance-encoding plasmid designated pCVD1010 (see Figure 2), contains an aroD gene harboring a 0.35 kb deletion, designated delaroD1013.
  • the EcoRI site present in the precise location of deletion delaroD1013 is flanked by 15 base pairs of non-coding and innocuous DNA from Tn1725 (see Figure 2).
  • the bla gene of pCVD1010 encoding ampicillin resistance, was then substituted by the cat gene, encoding chloramphenicol resistance, from pKTN701 (Nishibuchi, Pepartment of Microbiology, Faculty of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo, Kyoto 606, Japan) or any other well-known plasmid containing the cat gene, such as pACYC184 (New England Biolabs, Beverly, MA). This was achieved by substituting the 0.7 kb Pstl fragment of pCVD1010 with the 3.8 kb Pstl fragment of pKTN701 (see Figure 2).
  • plasmids pCVP1010 and pKTN701 were digested with Pstl and the enzyme was inactivated by placing the digested DNA at 65oC for 5 min. The digested plasmid DNAs were mixed and ligated. The resulting ligated DNA was transformed into SY327 and selection for resistance to 20 ⁇ g/ml of chloramphenicol was carried out. Chloramphenicol-resistant colonies were screened for the presence of plasmids with the appropriate restriction pattern. One such clone was selected for and the resulting plasmid was designated pCVD1013 (see Figure 2).
  • Plasmid pCVD1013 was subsequently used to introduce delaroD1013.
  • Cosmid pBRP138 obtained from Pr. G. Dougan, carries approximately 17 kb of the S. typhi chromosome and carries a functional aroC gene.
  • An 8.0 kb Bglll-generated fragment of pBRP138 was subcloned into BamHI-digested pUC9 (Yanisch-Perron et al, Gene, 33:103-107 (1985)) to create pCVD1003 (see Figure 3).
  • pBRP138 was digested with Bglll and pUC9 was digested with BamHI. Then, the restriction enzymes were inactivated as described above. The two digested plasmids were mixed and ligated and the ligated DNA was transformed into
  • Pstl and Nrul restriction sites both located within the aroC coding region, were employed (see Figure 3). More specifically, plasmid pCVD1003 was digested with SaIl, circularized by ligation and transformed into DH5- ⁇ (Bethesda Research Laboratories, Gaithersburg, MD). The resulting plasmid, designated pCVD1015, was found to lack the 1.0 kb SaIl fragment and had a unique Nrul site.
  • Plasmid pCVD1015 was digested with Nrul and the Nrul-generated ends were converted to Pstl ends by ligation to Pstl linkers (Bethesda Research Laboratories, Gaithersburg, MD). This modified plasmid DNA was then digested with Pstl, circularized by ligation and transformed into DH5- ⁇ . The resulting plasmid, designated pCVD1016, was found to have lost the 0.654 kb Nrul to Pstl fragment located within the aroC gene. This fragment was replaced by a Pstl site. Next, plasmid pCVD1016 was digested with EcoRI and SaIl and ligated to EcoRI- and Sail-digested pGP704.
  • the resulting plasmid designated pCVD1011, carries a 1.6 kb SaIl to EcoRI insert which in turn carries the deletion inactivated aroC gene designated delaroC1019 (see Figure 4), which is missing the 0.654 kb Nrul to Pstl fragment.
  • the bla gene of pCVD1011 was substituted by the cat gene of pKTN701 by ligating the 4.6 kb Sail to BamHI fragment of pKTN701 to the 2.1 kb SaIl to BamHI fragment of pCVD1011 (see Figure 4).
  • pCVD1011 was digested with SaIl and BamHI. Then, the 2.1 kb fragment was excised from an agarose gel after electrophoresis and purified. Plasmid pKTN701 also was digested with SaIl and BamHI and the enzymes were inactivate as described above. These two DNA preparations were mixed, then ligated and transformed into SY327 and selection for resistance to 20 ⁇ g/ml of chloramphenicol was carried out. Chloramphenicol-resistant colonies were screened for the presence of plasmids with the appropriate restriction pattern. One such clone was selected for and the resulting plasmid was designated pCVP1019 (see Figure 4).
  • Plasmid pCVD1019 was subsequently used to introduce delaroC1019, the deletion-inactivated aroC gene carried by pCVD1019, into the chromosome of S . typhi strain ISP1820.
  • Plasmid pCVD1017 carries a 0.654 kb Pstl to Nrul fragment of pCVD1003 which is located within the aroC coding region and which is deleted in delaroC1019-mutant strains.
  • pCVD1017 was constructed by digesting plasmid pCVD1003 with Pstl and Nrul. The 0.6 kb fragment flanked by Pstl- and Nrul-generated ends was then inserted by ligation into Smal- and Pstl-digested pUC19 (Yanisch-Perron et al, Gene, 33:103-107 (1985)) so as to obtain plasmid pCVD1017.
  • Plasmid pCVD1017 is useful as a probe because it does not hybridize with delaroC1019-mutant strains.
  • Plasmid pCVD1018 carries the 0.3 kb EcoRI to
  • pCVD1018 was constructed by digesting pCVD1006 with EcoRI and EcoRV. The 0.3 kb EcoRI to EcoRV fragment was isolated and ligated to EcoRI- and Smal-digested pUC19 DNA so as to obtain plasmid pCVD1018. Plasmid pCVD1018 is useful as a probe because it does not hybridize with delaroD1013-mutant strains. E. Introduction of delaroC1019 and delaroD1013 into the Chromosome of S. typhi ISP1820
  • Plasmids pCVD1013 and pCVD1019 cannot replicate in S. typhi.
  • pCVD1013 or pCVD1019 are introduced by electroporation into S . typhi they form stable cointegrates as a result of a single homologous recombination event between S. typhi DNA carried on the plasmids and the S. typhi chromosome.
  • Such cointegrates can be cured using a chloramphenicol-sensitive enrichment process.
  • a significant proportion of the chloramphenicol-sensitive derivatives of the cointegrate strains S. typhi::pCVD1019 or S. typhi::pCVD1013 carry the delaroC1019 mutation and the delaroD1013 mutation, respectively.
  • S. typhi strain ISP1820 (ISP1820 has been deposited at the American Culture Type Collection on May 15, 1990, under ATCC No. 55047) was transformed by electroporation with pCVD1019 (see Figure 5).
  • the electroporation procedure was a modification of the procedure described by Sambrook et al. In: Molecular Cloning a Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).
  • ISP1820 were taken from a fresh L-agar plate, placed into 10 ml of L-broth and incubated for 16 hr at 37oC with aeration and with shaking at 150 opm. Then, the 16 hr culture was diluted 1:100 into 250 ml of fresh L-broth in a 1.0 liter baffled flask and grown at 37oC with aeration and with shaking at 250 opm until the OD 600 reached 0.3 relative to an L-broth control. The cells were pelleted by centrifugation at 4000 x g at 4oC and then gently resuspended in 100 ml of cold, sterile, and ultra-pure H 2 O (Millipore milli-Q system).
  • the cells were pelleted again as described above and resuspended in another 100 ml of H 2 O. The 100 ml-H 2 O wash was repeated one more time. After one final pelleting, the cells were resuspended in 10% (v/v) glycerol to a cell density of 2 x 10 10 /ml. To a 400 ⁇ l aliquot of the cell suspension, 2.5 ⁇ g of plasmid pCVD1019 DNA, suspended in 10 ⁇ l of H 2 O, was added and the DNA/cell mixture was placed into a 0.2 cm cuvette (Bio-Rad, Richmond, CA).
  • the DNA/cell mixture was pulsed, typically with time decay factors of between 9.0 to 11.0 msec.
  • the cells were immediately transferred to 10 ml of SOC recovery medium comprising 1.2% (w/v) Bactone-tryptone (Pifco, Petroit, MI), 0.5% (w/v) Yeast extract (Sigma, St. Louis , MO) , 0.05% (w/v) NaCl (Sigma, St. Louis, MO) and 20 mM glucose (Sigma, St.
  • chloramphenicol-resistant control plasmid DNA pACYC184 (New England Biolabs, Beverly, MA; and Chang et al, J. Bacteriol., 134:1141-1156 (1978)), was introduced into in ISP1820.
  • Chloramphenicol-resistant derivatives were the result of pCVD1019 forming a cointegrate with the chromosome of ISP1820 at the aroC locus.
  • One such chloramphenicol-resistant cointegrate of ISP1820 was chosen for further derivation and was designated H236.1 (see Figure 5).
  • chromosomal DNA isolated from H236.1, was purified and probed with [ 32 P]-labelled pCVD1003. This demonstrates that H236.1 had an altered chromosomal restriction pattern consistent with pCVD1019 forming a cointegrate with the chromosome.
  • strain H236.1 was grown on L-agar containing 20 ⁇ g/ml of chloramphenicol at 37oC for 16 hr. Then, about 1 x 10 6 cells from this culture were transferred to Aro-broth comprising L-broth supplemented with 100 ⁇ g/ml of ferric ammonium citrate, 0.01% (w/v) PABA, 0.01% (w/v) PHB, 0.25% (w/v) glucose and 0.2% (w/v) casamino acids (pH 6.0), and incubated at 37oC with aeration and shaking at 150 opm. After 16 hr, the cells were diluted to an OD 600 of 0.05 relative to an Aro-broth control.
  • chloramphenicol was added to a concentration of 20 ⁇ g/ml and the culture was incubated at 37o C with aeration and with shaking at 250 opm until the OD 600 reached 0.2 relative to an Aro-broth control.
  • P-cycloserine Sigma, St. Louis, MO
  • P-cycloserine Sigma, St. Louis, MO
  • the chloramphenicol-sensitive bacteria which were static due to the chloramphenicol added at the beginning, remained unaffected by the P-cycloserine. Lysis was accompanied by a drop in OD 600. Cells were harvested by centrifugation at 4000 x g and gently resuspended in 10 ml of saline. The pelleting resuspension steps were repeated two more times. Then, 10-fold serial dilutions were made of this final cell suspension and 100 ⁇ l aliquots were spread evenly onto Aro-agar plates.
  • S. typhi strain ISP1820 and the mutants thereof were grown in Aro-broth at 37oC to an OP 600 of 0.6.
  • the cells were harvested by centrifugation at 4000 x g and resuspended in 10 ml of saline. After repeating the centrifugation step as described above, the cells were resuspended in saline. Then, 10-fold serial dilutions of the cell suspensions were mixed with 5.0% (w/v) hog gastric mucin (Wilson Laboratories, lot #0347A001) as described by Hone et al, Infect. Immun., 56:1326-1333 (1988). The bacterial dilutions suspended in 5.0% (w/v) hog gastric mucin were injected intraperitoneally into 18-20 g female CD-1 mice (Charles River, PA). The LP 50 values were calculated as described by Hone et al, Infect. Immun., 56:1326-1333 (1988). The results are shown in Table 6 below.
  • Typhi-M9 medium 0.01% (w/v) tryptophan, 0.1% (w/v) cysteine, and 100 ⁇ g/ml of ferric ammonium citrate
  • Typhi-M9 medium 0.01% (w/v) PABA, 0.01% (w/v) DHB, 0.01% (w/v) p-hydroxybenzoate, 10 ⁇ g/ml of ferric ammonium citrate, 0.01% (w/v) tyrosine and 0.01% (w/v) phenylalanine
  • M9+ARO medium The results are shown in Table 7 below.
  • strain CVD906 could mimic the growth of parent strain, ISP1820, in Typhi-M9 medium and human serum, but only if supplemented with 0.01% (w/v) PABA, 0.01% (w/v) DHB, 0.01% (w/v) p-hydroxybenzoate, 10 ⁇ g/ml of ferric ammonium citrate (serum only), 0.01% (w/v) tyrosine and 0.01% (w/v) phenylalanine; i.e., the nutrients required to complement the aro mutations.
  • acid-tolerant strain CVD906 At a dose of 3 x 10 7 viable organisms, acid-tolerant strain CVD906 impressively survived passage through the gastric barrier and was excreted in high titer for 2 to 4 days by all of the 9 volunteers and in 2 volunteers, excreted organisms reappeared again at day 10 or day 11 post-vaccination. Also, 5 of the volunteers produced positive blood cultures 6 to 7 days after vaccination. These two observations, taken together, indicate that the vaccine strain colonized the intestine far better than expected, thus enabling larger numbers of acid-tolerant strain CVD906 to invade through the mucosa and enter the systemic circulation. Eight of the volunteers tolerated the vaccine without fever, while 1 volunteer developed fever, anorexia, headache, bronchitic cough and abdominal discomfort. All of the isolates from the volunteers demonstrated the phenotypic characteristics of the vaccine strain. These include a negative H 2 S reaction and an inability to grow on Typhi-M9 medium that was not supplemented with aromatic amino acids, PABA and DHB.
  • Intestinal immunological priming by acid-tolerant strain CVD906 was measured using the antibody secreting cell (ASC) assay, carried out as described by Czerkinsky et al, J. Immunol. Meth., 65:109-121 (1983); and Czerkinsky et al, Proc. Natl. Acad. Sci. USA, 84:2449-2453 (1987). The results are shown in Table 8 below. For purposes of comparison, Table 8 below also shows the ASC responses to S. typhi lipopolysaccharide antigen in the 9 volunteers who have received a single 5 x 10 7 organism dose of acid-tolerant strain CVD906 versus the 9 volunteers who received three 10 9 organism doses of acid-sensitive strain Ty21a-Vi + .
  • ASC antibody secreting cell

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Abstract

L'invention se rapporte au bacille Salmonella typhi tolérant les acides et à des mutants aro doubles de ce bacille, qui sont utiles dans la préparation d'une seule dose de vaccin à administrer par voie buccale contre la fièvre typhoïde et comme vecteur de gènes qui expriment des antigènes protecteurs clonés à partir d'autres agents pathogènes. L'invention décrit également un procédé d'enrichissement du bacille Salmonella typhi tolérant les acides.
PCT/US1991/003447 1990-05-23 1991-05-22 Bacille salmonella typhi tolerant les acides, mutants aro doubles de ce bacille et utilisation de ce bacille comme vaccin administre par voie buccale contre la fievre typhoide WO1991018092A1 (fr)

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EP0564689A1 (fr) * 1992-04-10 1993-10-13 SCHWEIZERISCHES SERUM- & IMPFINSTITUT BERN Vaccin recombinant vivant contre des agents pathogène entériques Gram-négatifs
WO1998003661A2 (fr) * 1996-07-19 1998-01-29 Arch Development Corporation Agents antimicrobiens, reactifs de diagnostic et vaccins a base de composants specifiques du parasite apicomplexan
US5747028A (en) * 1993-02-22 1998-05-05 The President And Fellows Of Harvard College Immunizing compositions comprising Vibrio cholerae expressing heterologous antigens
WO1998026799A1 (fr) * 1996-12-18 1998-06-25 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vaccin conjugue contre la salmonella paratyphi a
US6020144A (en) * 1996-09-12 2000-02-01 Symbiontics, Inc. Sustained delivery device comprising a Leishmania protozoa and methods of making and using the same
US6036953A (en) * 1996-11-29 2000-03-14 The General Hospital Corporation Heterologous antigens in live cell V. cholerae strains
WO2000060102A2 (fr) * 1999-04-05 2000-10-12 Innovation And Development Corporation, University Of Victoria Systeme de pilus bacterien pour la presentation de sequences peptidiques heterologues

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INFECTION AND IMMUNITY, Volume 53, No. 6, issued September 1986, J.D. CLEMENTS, "Oral Immunization of Mice with Attenuated Salmonella Enteridis Containing a Recombinant Plasmid which Codes for Production of the B Subunit of Heat-Labile Escherichia Coli Enterotoxin", pages 685-692. *
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THE JOURNAL OF INFECTIOUS DISEASES, Volume 155, No. 1, issued January 1987, A. BROWN et al., "An Attenuated aroA Salmonella Typhimurium Vaccine Elicits Hormonal and Cellular Immunity to Cloned B-Galactosidase in Mice", pages 86-92. *
THE JOURNAL OF INFECTIOUS DISEASES, Volume 158, No. 6, issued December 1988, G. DOUGAN et al., "Construction and Characterization of Vaccine Strains of Salmonella Harboring Mutations in Two Different Aro Genes", pages 1329-1335. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0564689A1 (fr) * 1992-04-10 1993-10-13 SCHWEIZERISCHES SERUM- & IMPFINSTITUT BERN Vaccin recombinant vivant contre des agents pathogène entériques Gram-négatifs
US5747028A (en) * 1993-02-22 1998-05-05 The President And Fellows Of Harvard College Immunizing compositions comprising Vibrio cholerae expressing heterologous antigens
WO1998003661A2 (fr) * 1996-07-19 1998-01-29 Arch Development Corporation Agents antimicrobiens, reactifs de diagnostic et vaccins a base de composants specifiques du parasite apicomplexan
WO1998003661A3 (fr) * 1996-07-19 1998-10-08 Arch Dev Corp Agents antimicrobiens, reactifs de diagnostic et vaccins a base de composants specifiques du parasite apicomplexan
US6020144A (en) * 1996-09-12 2000-02-01 Symbiontics, Inc. Sustained delivery device comprising a Leishmania protozoa and methods of making and using the same
US6410250B1 (en) 1996-09-12 2002-06-25 Symbiontics, Inc. Sustained delivery device and methods of making and using the same
US6036953A (en) * 1996-11-29 2000-03-14 The General Hospital Corporation Heterologous antigens in live cell V. cholerae strains
WO1998026799A1 (fr) * 1996-12-18 1998-06-25 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vaccin conjugue contre la salmonella paratyphi a
WO2000060102A2 (fr) * 1999-04-05 2000-10-12 Innovation And Development Corporation, University Of Victoria Systeme de pilus bacterien pour la presentation de sequences peptidiques heterologues
WO2000060102A3 (fr) * 1999-04-05 2001-01-04 Innovation And Dev Corp Univer Systeme de pilus bacterien pour la presentation de sequences peptidiques heterologues

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