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WO2008025171A1 - Vaccins et procédés pour le traitement ou la prévention d'infections bactériennes par des espèces salmonella chez un sujet vertébré - Google Patents

Vaccins et procédés pour le traitement ou la prévention d'infections bactériennes par des espèces salmonella chez un sujet vertébré Download PDF

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Publication number
WO2008025171A1
WO2008025171A1 PCT/CA2007/001555 CA2007001555W WO2008025171A1 WO 2008025171 A1 WO2008025171 A1 WO 2008025171A1 CA 2007001555 W CA2007001555 W CA 2007001555W WO 2008025171 A1 WO2008025171 A1 WO 2008025171A1
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Prior art keywords
salmonella spp
subject
vaccine
composition
cell culture
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English (en)
Inventor
Brett B. Finlay
Brian Coombes
Yuling Li
Andrew Potter
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SASKATCHEWAN AS REPRESENTED BY VACCINE AND INFECTIOUS DISEASE ORGANIZATION, University of
University of Saskatchewan
University of British Columbia
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SASKATCHEWAN AS REPRESENTED BY VACCINE AND INFECTIOUS DISEASE ORGANIZATION, University of
University of Saskatchewan
University of British Columbia
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Priority to JP2009525882A priority Critical patent/JP2010501599A/ja
Priority to CA002662174A priority patent/CA2662174A1/fr
Publication of WO2008025171A1 publication Critical patent/WO2008025171A1/fr
Anticipated expiration legal-status Critical
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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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 invention relates to vaccines and methods for the treatment or prevention of Gram negative bacterial infection in a vertebrate subject such as Salmonella spp. bacterial infection.
  • Methods are provided for administering a bacterial agent to the vertebrate subject in an amount effective to reduce or eliminate the bacterial colonization and/or infection.
  • Methods for the treatment or prevention of Salmonella spp. infection in an avian species or a mammalian species are provided.
  • Salmonella enterica cause significant morbidity and mortality worldwide.
  • the Salmonella enterica serovars causing disease in humans are Typhi, Paratyphi A and B, and Typhimurium.
  • the Typhi and Paratyphi serovars are strict human pathogens and cause typhoid fever, while the non-typhoidal Salmonella enterica subspecies Typhimurium results primarily in gastroenteritis in humans.
  • Typhimurium is not a strict human pathogen and can infect a number of animal species, with animal-human transmission being a common, and well-recognized problem.
  • Cross-species infection usually results from contamination of agricultural products such as chicken. These organisms can also cause significant agricultural disease, such as gastroenteritis seen in cattle.
  • Salmonella vaccines have been developed for poultry and swine use. Most efforts have focused on single and double gene modification (or knockout) attenuated Salmonella vaccines.
  • the gene(s) targeted for elimination are usually SPI-I and SPI-2 or aromatic amino acid biosynthetic pathways.
  • Bacterial infection of a vertebrate subject such as avaian species or mammalian species.
  • Salmonella infection can present as gastroenteritis, enteric fever, a bacteremic syndrome, or focal disease.
  • Salmonella spp. possess two type III secretion systems (SPI-I) and (SPI-2) that secrete virulence factors out of the bacterial cells and into host cells. The type III secretion system encoded by Salmonella spp.
  • SPI-2 pathogenicity islands 1 and 2
  • SPI-2 pathogenicity islands 1 and 2
  • SPI-2 pathogenicity islands 1 and 2
  • SPM and SPI-2 have a discrete set of secreted effector proteins.
  • the invention features an isolated Salmonella spp. cell culture supernatant.
  • the invention generally relates to vaccines and methods for the prevention or treatment of Gram negative bacterial infection and bacterial carriage such as Salmonella spp. bacterial infection and Salmonella spp. bacterial carriage, in a vertebrate subject.
  • the invention provides a vaccine composition comprising an effective immunizing amount of an isolated Salmonella spp. cell culture supernatent and a pharmaceutically acceptable carrier, wherein said vaccine composition is effective in a vertebrate subject to reduce or eliminate Gram-negative bacterial infection.
  • the infection is Salmonella spp. infection.
  • the composition is further effective in reducing or eliminating Salmonella spp. bacterial carriage.
  • the composition is effective in reducing or eliminating both bacterial carriage and bacterial infection.
  • cell culture supernatent is produced under SPI-2 inducing conditions by the process of incubating the cell culture in media comprising minimal media supplemented with a low phosphate, low magnesium medium (LPM), wherein the LPM medium composition is 5 mM KCl, 7.5 mM (NH 4 ) 2 SO 4 , 0.5 niM K 2 SO 4 , 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3' , 80 mM 2- [N-morpholino]ethanesulfonic acid (MES), pH 5.8.
  • LPM medium composition is 5 mM KCl, 7.5 mM (NH 4 ) 2 SO 4 , 0.5 niM K 2 SO 4 , 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3' ,
  • the LPM medium composition is 5 mM KCl, 7.5 mM (NRt) 2 SO 4 , 0.5 mM K 2 SO 4 , 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3" , and 100 mM Tris-HCl, pH 7.0.
  • the vertebrate subject is a mammalian subject or an avian subject.
  • the avian subject is one day of age or older.
  • the avian subject is in ovo.
  • the vaccine comprises an immunological adjuvant.
  • the adjuvant can be an oil-in-water emulsion, a mineral oil and dimethyldioctadecylammonium bromide, or VSA3.
  • the VS A3 is present in the composition at a concentration of about 20% to about 40% (v/v).
  • the VSA3 is present in the composition at a concentration of about 30% (v/v).
  • the vaccine composition comprises one or more recombinant or purified antigens selected from the isolated Salmonella spp. cell culture supernatent.
  • the invention provides a method for preventing or treating a Gram negative bacterial infection in a vertebrate subject comprising, administering an isolated Salmonella spp.
  • the Salmonella spp. cell culture supernatent is produced under SPI-2 inducing conditions by the process of incubating the cell culture in media comprising minimal media supplemented with a low phosphate, low magnesium medium (LPM), wherein the LPM medium composition is 5 mM KCl, 7.5 mM (NH 4 ⁇ SO 4 , 0.5 mM K 2 SO 4 , 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3" , 80 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 5.8.
  • LPM medium composition is 5 mM KCl, 7.5 mM (NH 4 ⁇ SO 4 , 0.5 mM K 2 SO 4 , 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3
  • the LPM medium composition is 5 mM KCl, 7.5 mM (NH 4 ) 2 SO 4 , 0.5 mM K 2 SO 4 , 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3" , 100 mM Tris-HCl, pH. 7.0.
  • the infection is Salmonella spp. infection.
  • a protein or agent from the isolated Salmonella spp. cell culture supernatant is involved in Type III secretion systems.
  • the vertebrate subject is a mammalian subject or an avian subject.
  • the avian subject is one day of age or older. In other such aspects, the avian subject is in ovo.
  • the method comprises an immunological adjuvant.
  • the immunological adjuvant comprises an oil-in-water emulsion, a mineral oil and dimethyldioctadecylammonium bromide, or VS A3.
  • the VS A3 is present in the composition at a concentration of about 20% to about 40% (v/v).
  • the VSA3 is present in the composition at a concentration of about 30% (v/v).
  • the vaccine composition comprises one or more recombinant or purified antigens selected from the isolated Salmonella spp. cell culture supernatent.
  • the invention provides a method for eliciting an immunological response in a vertebrate subject against a Salmonella spp. bacterial infection comprising, administering isolated Salmonella spp. cell culture supernatant to the vertebrate subject in an amount effective to reduce or eliminate the bacterial infection.
  • a protein or agent from the isolated Salmonella spp. cell culture supernatant is involved in Type III secretion systems.
  • the vertebrate subject is a mammalian subject or an avian subject.
  • the avian subject is one day of age or older. In other such aspects, the avian subject is in ovo.
  • the present invention provides a method for reducing colonization of Salmonella spp. bacteria in a vertebrate subject comprising administering to the vertebrate subject a composition comprising a protein from an isolated Salmonella spp. cell culture supernatant involved in Type III secretion systems and an immunological adjuvant in an amount effective to reduce Salmonella spp. bacterial count in the vertebrate subject.
  • the reduction of colonization of Gram negative bacteria in the vertebrate subject further comprises reducing a risk of infectious transfer from the vertebrate subject.
  • the invention provides a method for reducing shedding of Salmonella spp. bacteria in a vertebrate subject comprising administering to the vertebrate subject a composition comprising a protein from an isolated Salmonella spp. cell culture supernatant involved in Type III secretion systems and an immunological adjuvant in an amount effective to reduce Salmonella spp. bacterial count in the vertebrate subject.
  • the reducition of shedding of Salmonella spp. bacteria in the vertebrate subject further comprises reducing a risk of infectious transfer from the vertebrate subject.
  • FIG. 1 Scale-up production of secreted proteins using fermentor cultures grown under SPI-2 culture conditions. This figure shows the concentration of cell culture supernatents (CCS), then probed with a SPI-2 effector (SseB), which is a readout of SPI-2 inducing conditions. "Neat” means the supernatants are not concentrated, and “cone” means the supernatant was concentrated (and therefore more SseB is present). Supernatants were grown in either a flask or in a fermentor.
  • CCS cell culture supernatents
  • SseB SPI-2 effector
  • FIG. 1 ⁇ ssaR sp mutant lacking SPI-2 type III secretion activity.
  • This figure shows CCS profiles from two different supernatants: one from wild type (SL1344), and one from a SPI-2 apparatus mutant (ssaR). Both supernatants were made under conditions that favor SPI-2 inducing conditions as described herein, then probed with antibodies to SseB. As expected, the SPI-2 mutant has no SseB in the supernatant, yet this formulation (Vacc2) still gave good protection in mice.
  • SL1344 wild type
  • ssaR SPI-2 apparatus mutant
  • FIG. 3 Reproducible fermentor-scale production of secreted proteins. This figure shows that fermentor runs of supernatants can overproduce the supernatents in a reproducible fashion.
  • FIG. 4 Ceca from vaccinated mice show protection from pathological atrophy, showing the mean plus or minus the error for the four samples. This figure shows very good vaccine "efficacy". If a Salmonella infection is strong, the cecal weight is low as it gets shrivelled and diseased. Larger cecal weight means a healthier outcome. Mice were vaccinated with four different vaccines, and then all challenged orally with Salmonella. Both Saline and Saline/adjuvant had active and high infections, and thus low cecal weight (i.e., no protection). Vaccines containing supernatant from the wild type or a SPI-2 mutant (ssaR) gave good protection, and thus had normal cecal weights.
  • ssaR SPI-2 mutant
  • Figure 5 Ceca from vaccinated mice show protection from pathological atrophy, with significance statistic shown. Each point represents a single animal. Similar to Figure 4.
  • FIG. 6 Systemic bacterial load is significantly reduced in vaccinated mice (spleen). Another readout of Salmonella disease is the number of Salmonella in the spleen (spleens from infected mice are harvested, ground up, plated and counted). The higher the number, the worse the disease. This figure shows that both saline, and saline with adjuvant do not protect against Salmonella numbers in the spleen, but WT and SPI-2 mutant CCS (both with adjuvant) lowers the number of Salmonella in spleen by about 3-4 logs (i.e., strong protection).
  • FIG. 7 Systemic bacterial load is significantly reduced in vaccinated mice (liver). This figure is similar to the spleen data shown in Figure 6. Liver counts usually mimic the spleen counts.
  • FIG. 8 Vaccinated mice showing ⁇ 51og reduction in cecal bacterial load. This figure is similar to Figures 6 and 7, except it shows the cecal counts (cecum is in the intestine) A drastic significant decrease in the number of bacteria in the intestine is shown, implying that the vaccine would reduce shedding.
  • Figure 9 Pathological scores in vaccinated and control mice following infection with Salmonella enterica serovar Typhimurium. A pathological scoring system for grading disease was employed. Each mouse received a score (4 mice for each vaccine). Figure 9 shows that CCS from WT and SPI-2 mutant have much less pathology (i.e., much less disease) following challenge.
  • Figure 10 Means of Pathological scores in vaccinated and control mice following infection with Salmonella enterica serovar Typhimurium. This figure shows the average pathological scores of Figure 9.
  • FIG. 11 Colonization of the ceca (A) and other organs (B) following infection with Salmonella Typhimurium. Chickens were vaccinated with supernatants from bacteria grown under SPI-I or SPI-2 inducing conditions. SPI-2 conditions were from UBC (flask grown) or NRC (Fermentor grown). This figure shows that CCS grown in SPI-2 inducing conditions decrease chicken colonization, but not CCS grown in SPI-I inducing conditions.
  • the present invention generally relates to vaccines and methods for the prevention or treatment of Gram negative bacterial infection and bacterial carriage such as Salmonella spp. bacterial infection and Salmonella spp. bacterial carriage, in a vertebrate subject.
  • Methods for the treatment or prevention of Salmonella spp. infection or carriagein an avian species or a mammalian species are provided.
  • the methods provide administering a protein or agent involved in Type III secretion systems to the vertebrate subject in an amount effective to reduce or eliminate the Gram negative bacterial infection or bacterial carriage (e.g., the Salmonella spp. bacterial infection or carriage or both).
  • Vaccine compositions and method are provided for reducing colonization of Gram negative bacteria, such as Salmonella spp. bacteria, in an avian species comprising administering to the avian species a composition comprising of an isolated Salmonella spp. cell culture supernatant or a Salmonella spp. cell culture supernatant protein or agent, involved in Type HI secretion systems and an immunological adjuvant in an amount effective to reduce Gram negative bacterial count in the avian species (such as Salmonella spp. bacterial count).
  • the method further comprises reducing a risk of infectious transfer from the avian (or bovine) species to humans.
  • Salmonella spp. cell culture supernatant or Salmonella spp. “CCS” refers to a supernatant derived from a cell culture of one or more Salmonella spp. serotypes, which supernatant is substantially free of Salmonella spp. bacterial cells or the lysate of such cells, and which contains a mixture of Salmonella spp. antigens that have been secreted into the growth media.
  • a Salmonella spp. "CCS” will contain at least the secreted antigens responsible for Salmonella spp. infection, and fragments or aggregates thereof.
  • the CCS of the present invention can also include other secreted proteins or agents.
  • the proteins can be present in a native form, or a denatured or degraded form, so long as the CCS still functions to stimulate an immune response in the host subject such that Salmonella spp. disease is lessened or prevented, and/or colonization of Salmonella spp. is lessened or suppressed.
  • a CCS can be supplemented with additional recombinant or purified secreted antigens, as well as with any of the other secreted proteins.
  • MPL Monophosphoryl - lipid A
  • TDM Trehalose dicorynomycolate
  • RIBI commercially available adjuvant, from Sigma
  • Sm Streptomycin
  • SP Secreted proteins
  • SPI Salmonella pathogenicity island
  • “Vertebrate,” “mammal,” “subject,” or “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, dogs, cows, avian species, ducks, geese, chickens, amphibians, and reptiles.
  • avian subjects refers to males and females of any avian species, but is primarily intended to encompass poultry which are commercially raised for eggs, meat or as pets. Accordingly, the terms “avian” and “avian subject” are particularly intended to encompass chickens, turkeys, ducks, geese, quail, pheasant, parakeets, parrots, and the like.
  • the avian subject can be a hatched bird, which term encompasses newly- hatched (i.e., about the first three days after hatch) as well as post-hatched birds such as, for example, adolescent, and adult birds.
  • vaccine refers to a CCS composition that serves to stimulate an immune response to an Salmonella spp. antigen, such as a type III secreted Salmonella spp. antigen, therein.
  • the immune response need not provide complete protection and/or treatment against Salmonella spp. infection or against colonization and shedding of Salmonella spp. Even partial protection against colonization and shedding of Salmonella spp. bacteria will find use herein as shedding and contaminated meat production will still be reduced.
  • a vaccine will include an immunological adjuvant in order to enhance the immune response.
  • adjuvant refers to an agent which acts in a nonspecific manner to increase an immune response to a particular antigen or combination of antigens, thus reducing the quantity of antigen necessary in any given vaccine, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. See, e.g., A. C. Allison J. Reticuloendothel. Soc. (1979) 26:619-630. Such adjuvants are described further below.
  • colonization refers to the presence of Gram negative bacteria, e.g., Salmonella spp., in the intestinal tract of a mammal, such as a ruminant.
  • shedding refers to the presence of Gram negative bacteria, e.g., Salmonella spp., in feces.
  • Bacterial carriage is the process by which bacteria such as Salmonella spp. and can thrive in a normal subject without causing the subject to get sick. Bacterial carriage is a very complex interaction of the environment, the host and the pathogen. Various factors dictate asymptomatic carriage versus disease. Therefore an aspect of the invention includes
  • Treating” or “treatment” refers to either (i) the prevention of infection or reinfection, e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of the disease of interest, e.g., therapy.
  • Treating” or “treatment” can refer to the administration of a vaccine composition comprising a protein antigen involved in Type III secretion systems (for example a Salmonella spp. CCS protein). Treating an avian or mammalian species with the vaccine composition can prevent or reduce the risk of infection to humans. Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • Treating” or “treatment” can also refer to the administration of an antibody compositions, compounds or agents, peptide, peptidomimetic, a small chemical inhibitor RNA, short hairpin RNA, ribozyme, or antisense oligonucleotide of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease resulting from Salmonella spp. bacterial infection or bacterial carriage, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder (e.g., Salmonella spp. bacterial infection or bacterial carriage).
  • an antibody compositions, compounds or agents, peptide, peptidomimetic, a small chemical inhibitor RNA, short hairpin RNA, ribozyme, or antisense oligonucleotide of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease resulting from Salmonella spp. bacterial infection or bacterial carriage, alleviating the symptoms
  • Preventing refers to prophylactic administration or vaccination with antigen formations, for example, an antigen comprising a protein involved in Type III secretion systems (for example a Salmonella spp. CCS protein).
  • Preventing infection with a Gram negative bacteria e.g., Salmonella spp., refers to preventing colonization of an avian or mammalian species. Morbidity or mortality can result from infection or colonization of an avian species or mammalian species.
  • Preventing can also refer to antibody compositions, compounds or agents, peptide, peptidomimetic, a small chemical inhibitor RNA, short hairpin RNA, ribozyme, or antisense oligonucleotide of the present invention prior to exposure of the vertebrate subject to the Salmonella spp. bacteria to prevent or significantly reduce the level of Salmonella spp. bacterial infection in the vertebrate subject.
  • “Therapeutically-effective amount” or “an amount effective to reduce or eliminate bacterial infection” refers to an amount of an antagonist of a Salmonella spp. CCS protein that is sufficient to prevent Gram- negative bacterial infection or to alleviate (e.g., mitigate, decrease, reduce) at least one of the symptoms associated with Salmonella spp. bacterial infection. It is not necessary that the administration of the compound eliminate the symptoms of Salmonella spp. bacterial infection, as long as the benefits of administration of compound outweigh the detriments. Likewise, the terms “treat” and “treating” in reference to Salmonella spp.
  • bacterial infection as used herein, are not intended to mean that the avian subject is necessarily cured of infection or that all clinical signs thereof are eliminated, only that some alleviation or improvement in the condition of the avian subject is effected by administration of the an antagonist of a protein or agent involved in Type III secretion systems.
  • a protein or agent involved in Type III secretion systems refers to a protein or agent such as a Salmonella spp. CCS protein that is involved in Type III secretion systems.
  • a protein involved in Type III secretion systems that is regulated by a bacterial Salmonella spp. CCS protein or agent refers to a protein that is a regulatory target of the Salmonella spp.CCS protein or agent.
  • Protective immunity or “protective immune response,” are intended to mean that the host mammal or host bird mounts an active immune response to an antigenic component of bacterial flagella, such that upon subsequent exposure to the Gram-negative bacteria or bacterial challenge, the mammal or bird is able to combat the infection. Thus, a protective immune response will decrease the incidence of morbidity and mortality from subsequent exposure to the Gram-negative bacteria among host mammals or host birds.
  • a protective immune response will also decrease colonization by the Gram- negative bacteria in the host birds. In this manner, transmission of infectious Gram-negative bacteria from avian species to mammalian species will be decreased and controlled.
  • a protective immune response can be assessed by evaluating the effects of vaccination on the flock as a whole, e.g., there can still be morbidity and mortality in individual vaccinated birds.
  • Active immune response refers to an immunogenic response of the subject to an antigen.
  • this term is intended to mean any level of protection from subsequent exposure to Gram-negative bacteria or bacterial antigens which is of some benefit in a population of subjects, whether in the form of decreased mortality, decreased lesions, improved feed conversion ratios, or the reduction of any other detrimental effect of the disease, and the like, regardless of whether the protection is partial or complete.
  • An "active immune response” or “active immunity” is characterized by "participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development cell-mediated reactivity, or both.” Herbert B.
  • an active immune response is mounted by the host after exposure to immunogens by infection, or as in the present case, by vaccination.
  • Active immunity can be contrasted with passive immunity, which is acquired through the "transfer or preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a nonimmune host.” Id.
  • Inhibitors include inhibitors and activators. Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate bacterial proteins involved in Type III secretion systems, e.g., antagonists.
  • Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of proteins involved in Type III secretion systems, e.g., agonists.
  • Modulators include agents that, e.g., alter the interaction of proteins involved in Type III secretion systems: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like.
  • Modulators include genetically modified versions of naturally-occurring proteins involved in Type III secretion systems, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Cell-based assays for inhibitors and activators include, e.g., applying putative modulator compounds to a cell expressing a Salmonella spp. CCS protein or agent and then determining the functional effects on Type III secretion systems, as described herein.
  • Cell based assays include, but are not limited to, in vivo tissue or cell samples from a mammalian subject or in vitro cell-based assays comprising proteins involved in Type III secretion systems that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples untreated with inhibitors
  • Inhibition of Type III secretion systems is achieved when the Type III activity value relative to the control is about 80%, optionally 50% or 25-0%.
  • Activation of Type III secretion systems is achieved when the Type III activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • the ability of a molecule to bind to proteins involved in Type III secretion systems can be determined, for example, by the ability of the putative ligand to bind to immunoadhesin (antibody to proteins involved in Type III secretion systems) coated on an assay plate. Specificity of binding can be determined by comparing binding to proteins not involved in Type III secretion systems.
  • Test compound refers to any compound tested as a modulator of proteins involved in Type III secretion systems.
  • the test compound can be any small organic molecule, or a biological entity, such as a protein, e.g., an antibody or peptide, a sugar, a nucleic acid, e.g., an antisense oligonucleotide, RNAi, or a ribozyme, or a lipid.
  • test compound can be modulators that are genetically altered versions of proteins involved in Type III secretion systems.
  • test compounds will be small organic molecules, peptides, lipids, or lipid analogs.
  • compositions comprise a mixture of Salmonella spp. secreted antigens.
  • the CCS of the present invention can also include other secreted proteins.
  • the CCS is supplemented with additional recombinant or purified Salmonella spp. antigens.
  • the compositions can comprise cell culture supernatants and additional adjuvants from more than one Salmonella serotype to provide protection against multiple Salmonella spp.
  • Organisms for example, the S. ente ⁇ ca serotype which includes S. enteritidis, S. typhimurium, and S. typhi.
  • Most nontyphoidal Salmonella infections are caused by S. enteritidis. These infections are common and remain a significant public health problem in the world.
  • Many serotypes of S. enteritidis have been given names and are referred to informally as if they were separate species even though they are not.
  • the most common Salmonella serotypes include S. typhimurium, S. enteritides and S. typhi.
  • Other serotypes include S. heidelberg, S. newport, S. javiana, S. oranienburg, S. muenchen, S.
  • a pharmaceutically acceptable adjuvant can be administered with the cell culture supernatant.
  • the compositions are administered in an amount effective to elicit an immune response to one or more of the secreted antigens, thereby reducing or eliminating Salmonella spp. infection. In some instances, Salmonella spp. colonization of the animal is reduced or eliminated.
  • the animal is a chicken or other bird. In other preferred aspects, the animal is a sheep or other ruminant. In other preferred aspects, the animal is human.
  • the cell culture supernatant is derived from a cell culture of S. enterica
  • Immunization with CCS stimulates the immune system of the immunized animal to produce antibodies against one or more secreted Salmonella spp. antigens, that block Salmonella spp. attachment to intestinal epithelial cells, interfere with Salmonella spp. colonization and, thereby, reduce Salmonella spp. shedding by the animal.
  • This reduction in Salmonella spp. shedding results in a reduction in Salmonella spp. contamination of food and water and a reduction in Salmonella spp. -caused disease in humans.
  • the unexpected and surprising ability of CCS immunization to prevent, reduce and eliminate Salmonella spp. colonization and shedding by mammals addresses a long-felt unfulfilled need in the medical arts, and provides an important benefit for humans.
  • the CCS of the present invention can be used to treat or prevent Salmonella spp. infections in other mammals such as humans. If used in humans, the CCS can be produced from a mutated Salmonella spp. which has been engineered to knock out or knock down toxicity.
  • the therapeutic effectiveness of CCS can be increased by adding thereto one or more of the secreted antigens in recombinant or purified form, fragments thereof and/or analogs thereof.
  • Other methods to increase the therapeutic effectiveness of CCS include, but are not limited to, complexing the CCS to natural or synthetic carriers and administering the CCS, before, at the same time as, or after another anti-Salmonella spp. agent.
  • agents include, but are not limited to, biological, biologically engineered, chemical, nucleic acid based and recombinant protein anti- Salmonella spp. agents.
  • the CCS for use herein can be obtained from cultures of any Salmonella spp. serotype, including, as disclosed herein. Such Salmonella spp. serotypes are readily obtained from sera of infected animals. Methods for isolated Salmonella spp. are well known in the art. Moreover, CCS can be obtained from Salmonella spp. serotypes that have been genetically engineered to knock-out or knock down toxicity.
  • CCS is produced by culturing Salmonella spp. bacteria in a suitable medium, under conditions that favor type III antigen secretion.
  • the type III secretion system encoded by Salmonella spp. pathogenicity islands 1 and 2 (SPI-2) are major virulence factors contributing to the pathogenesis of salmonellosis. Both SPI-I and SPI-2 have a discrete set of secreted effector proteins.
  • Suitable media and conditions for culturing Salmonella spp. bacteria are known in the art and described in, e.g., Coombes, B. K. et al, 2005, Proc. Natl. Acad. ScL U.S.A. 102: 17460-17465 and Coombes, B. K. et al, J Biol Chem. 279: 49804-49815 (incorporated herein by reference in their entireties).
  • CCS is produced by culturing Salmonella spp. bacteria in a suitable medium, under conditions that favor type III antigen secretion.
  • Preferred culture conditions for generating protective agents in the CCS are SPI-2 inducing conditions.
  • Suitable media and conditions for culturing Salmonella spp. bacteria are known in the art and described in e.g., Coombes, B. K. et al, 2005, Proc. Natl. Acad. ScL U.S.A. 102: 17460- 17465 and Coombes, B. K. et al.J Biol Chem. 279: 49804-49815 (incorporated herein by reference in their entireties).
  • LPM low phosphate, low magnesium medium
  • KCl 7.5 mM
  • NfLO 2 SO 4 0.5 rnM K 2 SO 4
  • 38 mM glycerol 0.1% casamino acids
  • 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3" 80 mM 2-[N- morpholino]ethanesulfonic acid (MES).
  • MES 2-[N- morpholino]ethanesulfonic acid
  • the LPM medium is at pH 7.0 and comprises 5 mM KCl, 7.5 mM (NH 4 ⁇ SO 4 , 0.5 mM K 2 SO 4 , 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3" , 100 mM Tris-HCl (for titration to pH 7.0).
  • the composition of LPM medium can be 5 mM KCl, 7.5 mM (NH 4 ) 2 SO 4 , 0.5 mM K 2 SO 4 , 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 ⁇ M MgCl 2 , 337 ⁇ M PO 4 3" , 100 mM Tris-HCl (for titration to pH 7.0), or 80 mM 2- [N-morpholino]ethanesulfonic acid (MES) (for titration to pH 5.8). Cultures can be grown at 37 0 C with shaking for 4-6 hours after which the optical density at 600 nm can be measured.
  • MES 2- [N-morpholino]ethanesulfonic acid
  • Bacteria can be collected by centrifugation for 2 minutes at 12,000 rpm (4 0 C).
  • the CCS can be passed through a filter, e.g., a 0.22 ⁇ m filter or other suitable filter known to one of skill in the art and then precipitated with trichloroacetic acid (10% final concentration, v/v) at 4 0 C for 4-16 hours.
  • bacteria from the cultures is separated from the culture supernatant and the culture supernatant can be concentrated, for example, using, for example, Tangential flow ultafiltration or any other suitable concentration method known to one of skill in the art.
  • nucleic acids used to practice this invention can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams, J. Am. Chem. Soc. 105: 661, 1983; Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994; Narang, Meth. Enzymol. 68: 90, 1979; Brown Meth. Enzymol. 68: 109, 1979; Beaucage, Tetra. Lett. 22: 1859, 1981; U.S. Pat. No. 4,458,066.
  • the invention provides oligonucleotides comprising sequences of the invention, e.g., subsequences of the exemplary sequences of the invention.
  • Oligonucleotides can include, e.g., single stranded poly-deoxynucleotides or two complementary polydeoxynucleotide strands which can be chemically synthesized.
  • nucleic acids such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook and Russell, ed., MOLECULAR CLONING: A LABORATORYMANUAL (3rd ED.), VoIs. 1-3, Cold Spring Harbor Laboratory, 2001; CURRENTPROTOCOLS MOLECULAR BIOLOGY; Ausubel, ed.
  • Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g.
  • fluid or gel precipitin reactions immunodiffusion, immuno-electrophoresis, adioimmunoassay (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
  • RIAs adioimmunoassay
  • ELISAs enzyme-linked immunosorbent assays
  • immuno-fluorescent assays Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
  • Obtaining and manipulating nucleic acids used to practice the methods of the invention can be done by cloning from genomic samples, and, if desired, screening and re-cloning inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
  • Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld, Nat. Genet.
  • MACs mammalian artificial chromosomes
  • yeast artificial chromosomes YAC
  • bacterial artificial chromosomes BAC
  • Pl artificial chromosomes see, e.g., Woon, Genomics 50: 306-316, 1998
  • Pl- derived vectors PACs
  • cosmids recombinant viruses, phages or plasmids.
  • the invention provides fusion proteins and nucleic acids encoding them.
  • a Salmonella spp. CCS protein can be fused to a heterologous peptide or polypeptide, such as N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
  • Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like.
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.).
  • metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle Wash.
  • the inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego CA) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification.
  • an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams, Biochemistry 34: 1787-1797, 1995; Dobeli, Protein Expr. Purif 12: 404-414, 1998).
  • the histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein.
  • a nucleic acid encoding a polypeptide is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof.
  • a promoter can be one motif or an array of nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • a "constitutive" promoter is a promoter which is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter which is under environmental or developmental regulation.
  • tissue specific promoter is active in certain tissue types of an organism, but not in other tissue types from the same organism.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • aspects of the invention provide expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the proteins of the invention.
  • Expression vectors and cloning vehicles can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), Pl-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast).
  • Vectors can include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.
  • nucleic acids of the invention can be cloned, if desired, into any of a variety of vectors using routine molecular biological methods; methods for cloning in vitro amplified nucleic acids are described, e.g., U.S. Pat. No. 5,426,039.
  • restriction enzyme sites can be "built into” a PCR primer pair.
  • the invention provides libraries of expression vectors encoding polypeptides and peptides of the invention. These nucleic acids can be introduced into a genome or into the cytoplasm or a nucleus of a cell and expressed by a variety of conventional techniques, well described in the scientific and patent literature. See, e.g., Roberts, Nature 328: 731, 1987; Schneider, Protein Expr. Purif. 6435: 10, 1995; Sambrook, Tijssen or Ausubel.
  • the vectors can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.
  • the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses which are stably or transiently expressed in cells (e.g., episomal expression systems).
  • Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences.
  • selection markers can code for episomal maintenance and replication such that integration into the host genome is not required.
  • the nucleic acids of the invention are administered in vivo for in situ expression of the peptides or polypeptides of the invention.
  • the nucleic acids can be administered as "naked DNA” (see, e.g., U.S. Pat. No. 5,580,859) or in the form of an expression vector, e.g., a recombinant virus.
  • the nucleic acids can be administered by any route, including peri- or intra-tumorally, as described below.
  • Vectors administered in vivo can be derived from viral genomes, including recombinantly modified enveloped or non- enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxyiridae, adenoviridiae, or picornnaviridiae. Chimeric vectors can also be employed which exploit advantageous merits of each of the parent vector properties (See e.g., Feng, Nature Biotechnology 15: 866-870, 1997). Such viral genomes can be modified by recombinant DNA techniques to include the nucleic acids of the invention; and can be further engineered to be replication deficient, conditionally replicating or replication competent.
  • vectors are derived from the adenoviral (e.g., replication incompetent vectors derived from the human adenovirus genome, see, e.g., U.S. Pat. Nos. 6,096,718; 6,110,458; 6,113,913; 5,631,236); adeno- associated viral and retroviral genomes.
  • Retroviral vectors can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof; see, e.g., U.S. Pat. Nos.
  • Adeno-associated virus (AAV)-based vectors can be used to adioimmun cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures; see, e.g., U.S. Pat. Nos. 6,110,456; 5,474,935; Okada, Gene Ther. 3: 957-964, 1996.
  • Expression cassette refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as a polypeptide of the invention) in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression can also be used, e.g., enhancers.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • operably linked indicates that the sequences are capable of effecting switch recombination.
  • expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
  • Vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non- episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding a polypeptide of the invention, or a vector of the invention.
  • the host cell can be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • Exemplary bacterial cells include E. coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.
  • Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art.
  • the vector can be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation.
  • Engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter can be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells can be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.
  • appropriate means e.g., temperature shift or chemical induction
  • Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.
  • Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.
  • the expressed polypeptide or fragment can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
  • Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines.
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides produced by host cells containing the vector can be glycosylated or can be non-glycosylated.
  • Polypeptides of the invention can or can not also include an initial methionine amino acid residue.
  • Cell-free translation systems can also be employed to produce a polypeptide of the invention.
  • Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof.
  • the DNA construct can be linearized prior to conducting an in vitro transcription reaction.
  • the transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.
  • the expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • nucleic acids encoding the polypeptides of the invention, or modified nucleic acids can be reproduced by, e.g., amplification.
  • the invention provides amplification primer sequence pairs for amplifying nucleic acids encoding polypeptides of the invention, e.g., primer pairs capable of amplifying nucleic acid sequences comprising the Salmonella spp. CCS protein, or subsequences thereof.
  • Amplification methods include, e.g., polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N. Y., 1990 and PCR STRATEGIES, 1995, ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4: 560, 1989; Landegren, Science 241: 1077, 1988; Barringer, Gene 89: 117, 1990); transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad.
  • LCR ligase chain reaction
  • the invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to an exemplary sequence of the invention, e.g., a Salmonella spp. CCS protein sequence, or the complement of any thereof, or a nucleic acid that encodes a polypeptide of the invention.
  • the stringent conditions are highly stringent conditions, medium stringent conditions or low stringent conditions, as known in the art and as described herein. These methods can be used to isolate nucleic acids of the invention.
  • nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of nucleic acid of the invention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or more residues in length, or, the full length of a gene or coding sequence, e.g., cDNA. Nucleic acids shorter than full length are also included.
  • nucleic acids can be useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA, antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites and the like.
  • a nucleic acid can be determined to be within the scope of the invention by its ability to hybridize under stringent conditions to a nucleic acid otherwise determined to be within the scope of the invention (such as the exemplary sequences described herein).
  • Stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but not to other sequences in significant amounts (a positive signal (e.g., identification of a nucleic acid of the invention) is about 10 times background hybridization). Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in e.g., Sambrook and Russell, ed., MOLECULAR CLONING: A LABORATORY MANUAL (3rd ED.), VoIs.
  • stringent conditions are selected to be about 5-10°C lower than the thermal melting point I for the specific sequence at a defined ionic strength pH.
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0 C for short probes (e.g., 10 to 50 nucleotides) and at least about 6O 0 C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide as described in Sambrook (cited above).
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5x SSC and 1% SDS incubated at 42° C or 5x SSC and 1% SDS incubated at 65° C, with a wash in 0.2x SSC and 0.1% SDS at 65° C.
  • a positive signal e.g., identification of a nucleic acid of the invention is about 10 times background hybridization.
  • Stringent hybridization conditions that are used to identify nucleic acids within the scope of the invention include, e.g., hybridization in a buffer comprising 50% formamide, 5x SSC, and 1% SDS at 42°C, or hybridization in a buffer comprising 5x SSC and 1% SDS at 65 0 C, both with a wash of 0.2x SSC and 0.1% SDS at 65°C.
  • genomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here.
  • Additional stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C. [0093] However, the selection of a hybridization format is not critical — it is the stringency of the wash conditions that set forth the conditions which determine whether a nucleic acid is within the scope of the invention.
  • Wash conditions used to identify nucleic acids within the scope of the invention include, e.g., a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 5O 0 C or about 55°C to about 60 0 C; or, a salt concentration of about 0.15 M NaCl at 72°C for about 15 minutes; or, a salt concentration of about 0.2X SSC at a temperature of at least about 50 0 C or about 55 0 C to about 60 0 C for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1X SSC containing 0.1% SDS at 68oC for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen and Ausubel for a description of SSC buffer and equivalent conditions.
  • the invention also provides nucleic acid probes for identifying nucleic acids encoding a polypeptide which is a modulator or inhibitor of Type III secretion systems or proteins, e.g., Salmonella spp. CCS protein, involved in Type III secretion systems.
  • the probe comprises at least 10 consecutive bases of a nucleic acid of the invention.
  • a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a nucleic acid of the invention.
  • the probes identify a nucleic acid by binding and/or hybridization.
  • the probes can be used in arrays of the invention, see discussion below.
  • the probes of the invention can also be used to isolate other nucleic acids or polypeptides.
  • the invention provides nucleic acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, for example, a Salmonella spp. CCS gene.
  • the invention provides polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a Salmonella spp. CCS protein.
  • the sequence identities can be determined by analysis with a sequence comparison algorithm or by a visual inspection. Protein and/or nucleic acid sequence identities (or homologies) can be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • sequence comparison of nucleic acids and proteins the BLAST and BLAST 2.2.2. or FASTA version 3.0t78 algorithms and the default parameters discussed below can be used.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol.
  • a preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the FASTA algorithm, which is described in Pearson & Lipman, Proc. Natl. Acad. ScL U.S.A. 85: 2444, 1988. See also Pearson, Methods Enzymol. 266: 227-258, 1996.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: //www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. U.S.A. 90: 5873- 5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. MoI. Evol. 35: 351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, Nuc. Acids Res. 12: 387-395, 1984.
  • Sequence identity refers to a measure of similarity between amino acid or nucleotide sequences, and can be measured using methods known in the art, such as those described below:
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least of at least 60%, often at least 70%, preferably at least 80%, most preferably at least 90% or at least 95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 bases or residues in length, more preferably over a region of at least about 100 bases or residues, and most preferably the sequences are substantially identical over at least about 150 bases or residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • “Homology” and “identity” in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.
  • sequence comparison one sequence can act as a reference sequence (an exemplary sequence of Salmonella spp.CCS gene product or polypeptide) to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the numbers of contiguous residues.
  • continugous residues ranging anywhere from 20 to the full length of an exemplary polypeptide or nucleic acid sequence of the invention, e.g., an isolated Salmonella spp.CCS polynucleotide or polypeptide, are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the reference sequence has the requisite sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to Salmonella spp.CCS polynucleotide or polypeptide, that sequence is within the scope of the invention.
  • Motifs which can be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
  • the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention.
  • the words "recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.
  • Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
  • the computer readable media can be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
  • Salmonella spp. CCS protein sequences can be used to design oligonucleotide probes and used to screen genomic or cDNA libraries for genes from other Salmonella spp. serotypes.
  • the basic strategies for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I, supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra; Sambrook et al, supra.
  • a clone from the screened library has been identified by positive hybridization, it can be confirmed by restriction enzyme analysis and DNA sequencing that the particular library insert contains an Salmonella spp. CCS gene, or a homolog thereof.
  • the genes can then be further isolated using standard techniques and, if desired, PCR approaches or restriction enzymes employed to delete portions of the full-length sequence.
  • genes can be isolated directly from bacteria using known techniques, such as phenol extraction and the sequence further manipulated to produce any desired alterations. See, e-g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.
  • DNA sequences encoding the proteins of interest can be prepared synthetically rather than cloned.
  • the DNA sequences can be designed with the appropriate codons for the particular amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292: 756; Nambair et al. (1984) Science 223: 1299; Jay et al. (1984) J. Biol. Chem. 259: 6311.
  • coding sequences for the desired proteins can be cloned into any suitable vector or replicon.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage ⁇ (E. col ⁇ ), pBR322 (E. col ⁇ ), pACYC177 (E. col ⁇ ), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl (gram- negative bacteria), pM ⁇ 290 (non-E. coli gram-negative bacteria), pHV14 (E.
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence can or can not contain a signal peptide or leader sequence.
  • Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397.
  • regulatory sequences can also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements can also be present in the vector, for example, enhancer sequences.
  • control sequences and other regulatory sequences can be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • a vector such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Mutants or analogs can be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et al, supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
  • the expression vector is then used to transform an appropriate host cell.
  • mammalian cell lines include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, as well as others.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • baby hamster kidney (BHK) cells baby hamster kidney (BHK) cells
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • MDBK Madin-Darby bovine kidney
  • bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs.
  • Yeast hosts useful in the present invention include, but are not limited to, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.
  • Insect cells for use with baculovirus expression vectors include, but are not limited to, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera fingiperda, and Trichoplusia ni.
  • the proteins of the present invention are produced by culturing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • Salmonella spp. CCS protein can also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M.
  • compositions of the present invention are formulated into compositions for delivery to a mammalian subject.
  • the vaccine composition is administered alone, or mixed with a pharmaceutically acceptable vehicle or excipient.
  • Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants in the case of vaccine compositions, which enhance the effectiveness of the vaccine. Suitable adjuvants are described further below.
  • the compositions of the present invention can also include ancillary substances, such as pharmacological agents, cytokines, or other biological response modifiers.
  • Vaccine compositions in aspects of the present invention can include adjuvants to further increase the immunogenicity of one or more of the Salmonella spp. CCS protein antigens.
  • adjuvants include any compound or compounds that act to increase an immune response to an Salmonella spp. CCS protein antigen or combination of antigens, thus reducing the quantity of antigen necessary in the vaccine, and/or the frequency of injection necessary in order to generate an adequate immune response.
  • Adjuvants can include for example, emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such as aluminum hydroxide, chitosan-based adjuvants, and any of the various saponins, oils, and other substances known in the art, such as Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, synthetic oligonucleotides and combinations thereof (Schijns et al, Curr. OpL Immunol. (2000) 12: 456), Mycobacterialphlei (M. phle ⁇ ) cell wall extract (MCWE) (U.S. Patent No. 4,744,984), M.
  • emulsifiers muramyl dipeptides, avridine
  • aqueous adjuvants such as aluminum hydroxide, chitosan-based adjuvants, and any of the various saponins, oils, and other substances known in the art, such as Amphigen, LPS,
  • phlei DNA M-DNA
  • M-DNA-M phlei cell wall complex MC
  • compounds which can serve as emulsifiers herein include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds.
  • anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of lauric and oleic acid, the calcium, magnesium and aluminum salts of fatty acids ⁇ i.e., metallic soaps), and organic sulfonates such as sodium lauryl sulfate.
  • Synthetic cationic agents include, for example, cetyltrhethylammonlum bromide, while synthetic nonionic agents are exemplified by glycerylesters ⁇ e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers, and the sorbitan fatty acid esters ⁇ e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives ⁇ e.g., polyoxyethylene sorbitan monopalmitate).
  • Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol.
  • suitable adjuvants can be formed with an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion.
  • the oil can be a mineral oil, a vegetable oil, or an animal oil.
  • Mineral oil, or oil-in-water emulsions in which the oil component is mineral oil are preferred.
  • a “mineral oil” is defined herein as a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique; the term is synonymous with “liquid paraffin,” “liquid petrolatum” and “white mineral oil.”
  • the term is also intended to include "light mineral oil,” i.e., an oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, supra.
  • a particularly preferred oil component is the oil-in-water emulsion sold under the trade name of EMULSIGEN PLUSTM (comprising a light mineral oil as well as 0.05% formalin, and 30 mcg/mL gentamicin as preservatives), available from MVP Laboratories, Ralston, California.
  • Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange roughy oil and shark liver oil, all of which are available commercially.
  • Suitable vegetable oils include, without limitation, canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like.
  • aliphatic nitrogenous bases can be used as adjuvants with the vaccine formulations.
  • known immunologic adjuvants include mines, quaternary ammonium compounds, guanidines, benzamidines and thiouroniums (Gall, D. (1966) Immunology 11: 369-386).
  • Specific compounds include dimethyldioctadecylammoniumbromide (DDA) (available from Kodak) and N,N- dioctadecyl-N,N-bis(2-hydroxyethyl)propanediine ("avridine").
  • DDA dimethyldioctadecylammoniumbromide
  • avridine N,N- dioctadecyl-N,N-bis(2-hydroxyethyl)propanediine
  • DDA immunologic adjuvant
  • Avridine is also a well-known adjuvant. See, e.g., U.S. Patent No.
  • VSA3 is a modified form of the EMULSIGEN PLUSTM adjuvant which includes DDA (see, U.S. Patent No. 5,951,988, incorporated herein by reference in its entirety).
  • Vaccine compositions including one or more of the Salmonella spp. CCS protein antigens in aspects of the present invention can be prepared by uniformly and intimately bringing into association the vaccine composition preparations and the adjuvant using techniques well known to those skilled in the art including, but not limited to, mixing, sonication and microfluidation.
  • the adjuvant will preferably comprise about 10 to 50% (v/v) of the vaccine, more preferably about 20 to 40% (v/v) and most preferably about 20 to 30% or 35% (v/v), or any integer within these ranges.
  • compositions of aspects of the present invention are normally prepared as injectables, either as liquid solutions or suspensions, or as solid forms which are suitable for solution or suspension in liquid vehicles prior to injection.
  • the preparation can also be prepared in solid form, emulsified or the active ingredient encapsulated in liposome vehicles or other particulate carriers used for sustained delivery.
  • the vaccine can be in the form of an oil emulsion, water in oil emulsion, water-in-oil-in-water emulsion, site- specific emulsion, long-residence emulsion, stickyemulsion, microemulsion, nanoemulsion, liposome, microparticle, microsphere, nanosphere, nanoparticle and various natural or synthetic polymers, such as nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures, that allow for sustained release of the vaccine.
  • nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers
  • swellable polymers such as hydrogels
  • resorbable polymers such as collagen and certain polyacids or polyesters such
  • the vaccine compositions including, for example, one or more of the Salmonella spp. CCS protein or agent antigens can be formulated into compositions in either neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the composition is formulated to contain an effective amount of a Salmonella spp. CCS protein or agent antigen, the exact amount being readily determined by one skilled in the art, wherein the amount depends on the animal to be treated and the capacity of the animal's immune system to synthesize antibodies.
  • the composition or formulation to be administered will contain a quantity of one or more secreted Salmonella spp. CCS protein or agent antigens adequate to achieve the desired state in the subject being treated.
  • a therapeutically effective amount of a vaccine comprising a cell extract of Salmonella spp. CCS protein or agent antigen with or without added recombinant and/or purified secreted Salmonella spp.
  • CCS protein or agent antigens contains about 0.05 to 1500 ⁇ g secreted protein or agent antigen, preferably about 10 to 1000 ⁇ g secreted protein or agent antigen, more preferably about 30 to 500 ⁇ g and most preferably about 40 to 300 pg, or any integer between these values.
  • Salmonella spp. CCS protein or agent antigen can comprise about 10% to 50% of total cell extract protein or agent, such as about 15% to 40% and most preferably about 15% to 25%. If supplemented with recombinant Salmonella spp. CCS protein or agent antigens, the vaccine can contain about 5 to 500 ⁇ g of protein or agent, more preferably about 10 to 250 ⁇ g and most preferably about 20 to 125 ⁇ g.
  • Routes of administration include, but are not limited to, oral, topical, subcutaneous, intramuscular, intravenous, subcutaneous, intradermal, transdermal and subdermal.
  • the volume per dose is preferably about 0.001 to 10 ml, more preferably about 0.01 to 5 ml, and most preferably about 0.1 to 3 ml.
  • Vaccine can be administered in a single dose treatment or in multiple dose treatments (boosts) on a schedule and over a time period appropriate to the age, weight and condition of the subject, the particular vaccine formulation used, and the route of administration.
  • any suitable pharmaceutical delivery means can be employed to deliver the compositions to the vertebrate subject, e.g., an avian subject or mammalian subject.
  • suitable pharmaceutical delivery means can be employed to deliver the compositions to the vertebrate subject, e.g., an avian subject or mammalian subject.
  • conventional needle syringes, spring or compressed gas (air) injectors U.S. Patent Nos. 1,605,763 to Smoot; 3,788,315 to Laurens; 3,853,125 to Clark et al.; 4,596,556 to Morrow et al; and 5,062,830 to Dunlap
  • liquid jet injectors U.S. Patent Nos. 2,754,818 to Scherer; 3,330,276 to Gordon; and 4,518,385 to Lindcaner et al
  • particle injectors U.S. Patent Nos. 5,149,655 to McCabe et al. and 5,204,253 to Sanford et al
  • a jet injector is used, a single jet of the liquid vaccine composition is ejected under high pressure and velocity, e.g., 1200-1400 PSI, thereby creating an opening in the skin and penetrating to depths suitable for immunization.
  • high pressure and velocity e.g. 1200-1400 PSI
  • An aspect of the invention relates to methods for preventing or treating in a subject a Salmonella spp. bacterial infection or bacterial carriage or both by administering vaccine composition comprising an effective immunizing amount a protein or agent involved Type III secretion systems is and a pharmaceutically acceptable carrier, wherein said vaccine composition is effective in a vertebrate subject to reduce or eliminate Gram- negative bacterial infection.
  • the therapeutic composition can also be an antagonist of Type III secretion systems administered to the vertebrate subject in an amount effective to reduce or eliminate the bacterial infection or bacterial carriage..
  • bacterial infection and bacterial carriage can be identified by, for example, any of a combination of diagnostic or prognostic assays as described herein or are known in the art.
  • such disorders involve undesirable activation of the innate immune system, e.g., as a result of Salmonella spp. bacterial infection or bacterial carriage.
  • Administration of the agent as a prophylactic agent can occur prior to the manifestation of symptoms, such that the symptoms are prevented, delayed, or diminished compared to symptoms in the absence of the agent.
  • the agent decreases Type III secretion systems.
  • the agent decreases synthesis or activity of proteins, e.g., Salmonella spp.CCS protein or agent, involved in Type III secretion systems.
  • the appropriate agent can be identified based on screening assays described herein. In general, such agents specifically bind to Salmonella spp.CCS polypeptide.
  • An aspect of the invention relates to methods for preventing or treating in a subject a Salmonella spp. bacterial infection or bacterial carriage by administering vaccine composition comprising an effective immunizing amount of a protein or agent involved in Type III secretion systems and a pharmaceutically acceptable carrier, wherein said vaccine composition is effective in a vertebrate subject to reduce or eliminate Gram-negative bacterial infection.
  • the present invention provides methods for preventing or treating an individual affected by a disease or disorder, e.g., Gram negative bacterial infection and more specifically, a Salmonell spp. bacterial infection or bacterial carriage.
  • the method involves administering a vaccine composition comprising an effective immunizing amount of an isolated Salmonella spp. CCS protein or agent involved in Type III secretion systems and a pharmaceutically acceptable carrier, or a vaccine composition comprising a therapeutic agent such as an inhibitor of proteins or agents, involved in Type III secretion systems.
  • Conditions that can be treated by agents include those in which a subject is treated for Gram negative bacterial infection, e.g., Salmonella spp. infection.
  • kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, and polypeptides.
  • the kits also can contain instructional material teaching the methodologies and uses of the invention, as described herein.
  • the compounds and modulators identified by the methods of the present invention can be used in a variety of methods of treatment.
  • the present invention provides compositions and methods for treating Gram negative bacterial infection such as Salmonella spp. infection, and Gram negative bacterial carriage, such as Salmonella spp. carriage.
  • infectious disease include but are not limited to, bacterial diseases.
  • the polypeptide or polynucleotide of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases can be treated.
  • the immune response can be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • the polypeptide or polynucleotide of the present invention can also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • Exemplary infectious disease include but are not limited to, Gram negative infections.
  • Gram-negative bacterial agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following Gram-negative bacterial families.
  • Bacteremia can be caused by Gram-negative bacteria.
  • Gram-negative bacteria have thin walled cell membranes consisting of a single layer of peptidoglycan and an outer layer of lipopolysacchacide, lipoprotein, and phospholipid.
  • Exemplary Gram-negative organisms include, but are not limited to, Enterobacteriacea consisting of Campylobacter, Escherichia, Shigella, Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter, Hafnia, Serratia, Proteus, Morganella, Providencia, Yersinia, Erwinia, Buttlauxella, Cedecea, Ewingella, Kluyvera, Tatumella and Rahnella.
  • Gram-negative organisms not in the family Enterobacteriacea include, but are not limited to, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia, Cepacia, Gardenerella, Vaginalis, and Acinetobacter species.
  • treatment using vaccine composition or a polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the mammalian or avian patient having a Gram negative bacterial infection or at risk for a Gram negative bacterial infection (for example, Salmonella spp. bacterial infection).
  • the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against Gram negative bacterial infectious disease, e.g., a Salmonella spp. infectious disease.
  • An aspect of the invention provides vaccine composition comprising an effective immunizing amount of a of an isolated Salmonella spp. cell culture supernatent protein or agent involved inType III secretion systems and a pharmaceutically acceptable carrier, wherein said vaccine composition is effective in a vertebrate subject to reduce or eliminate Gram-negative bacterial infection.
  • a further aspect provides pharmaceutical compositions comprising nucleic acids, polypeptides (including antibodies), peptidomimetic, small non-nucleic acid organic molecule, or small inorganic molecule of the invention. As discussed above, the nucleic acids, polypeptides, small chemical molecule of the invention can be used to inhibit expression of an endogenous Salmonella spp.CCS polypeptide.
  • Such inhibition in a cell or a non-human animal can generate a screening modality for identifying compounds to treat or ameliorate a Gram negative bacterial infection such as Salmonella spp. bacterial infection or Gram negative bacterial carriage.
  • Administration of a pharmaceutical composition of the invention to a subject is used to generate a toleragenic immunological environment in the subject. This can be used to tolerize the subject to an antigen.
  • the vaccine compositions, or nucleic acids, polypeptides, or small chemical molecule can be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition.
  • Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rates of the pharmaceutical compositions of the invention.
  • Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the peptides or polypeptides, or excipients or other stabilizers and/or buffers.
  • Detergents can also used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers.
  • Pharmaceutically acceptable carriers and formulations for peptides and polypeptide are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. ("Remington's").
  • physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, e.g., phenol and ascorbic acid.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the peptide or polypeptide of the invention and on its particular physio-chemical characteristics.
  • a solution of the vaccine composition or nucleic acids, peptides or polypeptides are dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier if the composition is water-soluble.
  • a pharmaceutically acceptable carrier e.g., an aqueous carrier if the composition is water-soluble.
  • aqueous solutions that can be used in formulations for enteral, parenteral or transmucosal drug delivery include, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions and the like.
  • the formulations can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.
  • Additives can also include additional active ingredients such as bactericidal agents, or stabilizers.
  • the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate.
  • These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered.
  • the resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • concentration of peptide in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • Solid formulations can be used for enteral (oral) administration. They can be formulated as, e.g., pills, tablets, powders or capsules.
  • conventional nontoxic solid carriers can be used which include, e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10% to 95% of active ingredient (e.g., peptide).
  • a non-solid formulation can also be used for enteral administration.
  • the carrier can be selected from various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • suitable pharmaceutical excipients include e.g., starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol.
  • Vaccine compositions or nucleic acids, polypeptides, or small chemical molecules when administered orally, can be protected from digestion. This can be accomplished either by complexing the nucleic acid, peptide or polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the nucleic acid, peptide or polypeptide in an appropriately resistant carrier such as a liposome.
  • Means of protecting compounds from digestion are well known in the art, see, e.g., Fix, Pharm Res. 13: 1760-1764, 1996; Samanen, /. Pharm. Pharmacol. 48: 119-135, 1996; U.S. Pat. No. 5,391,377, describing lipid compositions for oral delivery of therapeutic agents (liposomal delivery is discussed in further detail, infra).
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated can be used in the formulation.
  • penetrants are generally known in the art, and include, e.g., for transmucosal administration, bile salts and fusidic acid derivatives.
  • detergents can be used to facilitate permeation.
  • Transmucosal administration can be through nasal sprays or using suppositories. See, e.g., Sayani, Crit. Rev. Ther. Drug Carrier Sy st. 13: 85-184, 1996.
  • the agents are formulated into ointments, creams, salves, powders and gels.
  • Transdermal delivery systems can also include, e.g., patches.
  • Vaccine compositions or nucleic acids, polypeptides, or small chemical molecule as aspects of the invention can also be administered in sustained delivery or sustained release mechanisms, which can deliver the formulation internally.
  • sustained delivery or sustained release mechanisms can deliver the formulation internally.
  • biodegradeable microspheres or capsules or other biodegradeable polymer configurations capable of sustained delivery of a peptide can be included in the formulations of the invention (see, e.g., Putney, Nat. Biotechnol. 16: 153-157, 1998).
  • vaccine compositions or nucleic acids,nucleic acids, polypeptides, or small chemical molecule as aspects of the invention can be delivered using any system known in the art, including dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like. See, e.g., Patton, Biotechniques 16: 141-143, 1998; product and inhalation delivery systems for polypeptide macromolecules by, e.g., Dura Pharmaceuticals (San Diego, Calif.), Aradigrn (Hayward, Calif.), Aerogen (Santa Clara, Calif.), Inhale Therapeutic Systems (San Carlos, Calif.), and the like.
  • the pharmaceutical formulation can be administered in the form of an aerosol or mist.
  • the formulation can be supplied in finely divided form along with a surfactant and propellant.
  • the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes.
  • Other liquid delivery systems include, e.g., air jet nebulizers.
  • Avian subjects can be administered the vaccine compositions or nucleic acids, polypeptides, or small chemical molecule as aspects of the present invention by any suitable means.
  • exemplary means are oral administration (e.g., in the feed or drinking water), intramuscular injection, subcutaneous injection, intravenous injection, intraabdominal injection, eye drop, or nasal spray.
  • Avian subjects can also be administered the compounds in a spray cabinet, i.e., a cabinet in which the birds are placed and exposed to a vapor containing vaccine, or by coarse spray.
  • a spray cabinet i.e., a cabinet in which the birds are placed and exposed to a vapor containing vaccine, or by coarse spray.
  • administration by subcutaneous injection or spray cabinet are commonly used techniques.
  • the vaccine compositions or nucleic acids polypeptides, or small chemical molecule as aspects of the present invention can also be administered in ovo.
  • the in ovo administration of the compounds involves the administration of the compounds to the avian embryo while contained in the egg.
  • the compounds can be administered to any suitable compartment of the egg (e.g., allantois, yolk sac, amnion, air cell, or into the avian embryo itself), as would be apparent to one skilled in the art.
  • Eggs administered the compounds can be fertile eggs which are preferably in the last half, and more preferably the last quarter, of incubation. Chicken eggs are preferably treated on about day 18 of incubation, although other time periods can be employed.
  • the present invention can be carried out at various predetermined times in ovo.
  • Eggs can be administered the vaccine compositions or nucleic acids, polypeptides, or small chemical molecule of the invention by any means which transports the compound through the shell.
  • a common method of administration is, however, by injection.
  • the compound can injected into an extraembryonic compartment of the egg (e.g., yolk sac, amnion, allantois, air cell) or into the embryo itself.
  • the site of injection can be within the region defined by the amnion, including the amniotic fluid and the embryo itself.
  • the mechanism of egg injection is not critical, but it is preferred that the method not unduly damage the tissues and organs of the embryo or the extraembryonic membranes surrounding it so that the treatment will not decrease hatch rate.
  • the size of the needle and the length of penetration can be determined by one skilled in the art.
  • a pilot hole can be punched or drilled through the shell prior to insertion of the needle to prevent damaging or dulling of the needle.
  • the egg can be sealed with a substantially bacteria- impermeable sealing material such as wax or the like to prevent subsequent entry of undesirable bacteria.
  • compositions of the invention in vesicles composed of substances such as proteins, lipids (for example, liposomes, see below), carbohydrates, or synthetic polymers (discussed above).
  • lipids for example, liposomes, see below
  • carbohydrates for example, liposomes, see below
  • synthetic polymers discussed above.
  • Vaccine compositions or nucleic acids, polypeptides, or small chemical molecule of the invention can be delivered alone or as pharmaceutical compositions by any means known in the art, e.g., systemically, regionally, or locally (e.g., directly into, or directed to, a tumor); by intraarterial, intrathecal (IT), intravenous (IV), parenteral, intrapleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa).
  • one mode of administration includes intra-arterial or intrathecal (IT) injections, e.g., to focus on a specific organ, e.g., brain and CNS (see e.g., Gurun, Anesth Analg. 85: 317-323, 1997).
  • I intra-arterial or intrathecal
  • a specific organ e.g., brain and CNS
  • intra-carotid artery injection if preferred where it is desired to deliver a nucleic acid, peptide or polypeptide of the invention directly to the brain.
  • Parenteral administration is a preferred route of delivery if a high systemic dosage is needed.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in detail, in e.g., Remington's, See also, Bai, J. Neuroimmunol. 80: 65-75, 1997; Warren, J. Neurol. ScL 152: 31-38, 1997; Tonegawa, J. Exp. Med. 186: 507-515, 1997.
  • the pharmaceutical formulations comprising vaccine compositions or nucleic acids, polypeptides, or small chemical molecule of the invention are incorporated in lipid monolayers or bilayers, e.g., liposomes, see, e.g., U.S. Pat. Nos. 6,110,490; 6,096,716; 5,283,185; 5,279,833.
  • aspects of the invention also provide formulations in which water soluble nucleic acids, peptides or polypeptides of the invention have been attached to the surface of the monolayer or bilayer.
  • peptides can be attached to hydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing liposomes (see, e.g., Zalipsky, Bioconjug. Chem. 6: 705-708, 1995).
  • Liposomes or any form of lipid membrane such as planar lipid membranes or the cell membrane of an intact cell, e.g., a red blood cell, can be used.
  • Liposomal formulations can be by any means, including administration intravenously, transdermally (see, e.g., Vutla, J. Ph ⁇ rm. Sci. 85: 5-8, 1996), transmucosally, or orally.
  • the invention also provides pharmaceutical preparations in which the nucleic acid, peptides and/or polypeptides of the invention are incorporated within micelles and/or liposomes (see, e.g., Suntres, J. Ph ⁇ rm. Pharmacol. 46: 23-28, 1994; Woodle, Pharm. Res. 9: 260-265, 1992).
  • Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art, see, e.g., Remington's; Akimaru, Cytokines MoI. Ther. 1: 197-210, 1995; Alving, Immunol. Rev. 145: 5-31, 1995; Szoka, Ann. Rev. Biophys. Bioeng. 9: 467, 1980, U.S. Pat. Nos. 4, 235,871, 4,501,728 and 4,837,028.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/EDso.
  • Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 5O with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models, e.g., of inflammation or disorders involving undesirable inflammation, to achieve a circulating plasma concentration range that includes the IC 5 0 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • Levels in plasma can be measured, for example, by high performance liquid chromatography, generally of a labeled agent.
  • Animal models useful in studies, e.g., preclinical protocols, are known in the art, for example, animal models for inflammatory disorders such as those described in Sonderstrup (Springer, Sent. Immunopathol. 25: 35-45, 2003) and Nikula et al, Inhal. Toxicol. 4(12): 123-53, 2000), and those known in the art, e.g., for Gram-negative bacterial infection, e.g., Salmonella spp. infection.
  • a therapeutically effective amount of vaccine compositions, protein or polypeptide such as an antibody ranges from about 0.001 to 30 mg/kg body weight, for example, about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, or about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • the protein or polypeptide can be administered one or several times per day or per week for between about 1 to 10 weeks, for example, between 2 to 8 weeks, between about 3 to 7 weeks, or about 4, 5, or 6 weeks. In some instances the dosage can be required over several months or more.
  • treatment of a subject with a therapeutically effective amount of an agent such as a protein or polypeptide (including an antibody) can include a single treatment or, preferably, can include a series of treatments.
  • the dosage is generally 0.1 mg/kg of body weight (for example, 10 mg/kg to 20 mg/kg).
  • Partially human antibodies and fully human antibodies generally have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible.
  • Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain).
  • a method for lipidation of antibodies is described by Cruikshank et al., J. Acquired Immune Deficiency Syndromes and Human Retrovirology, 14: 193, 1997).
  • aspects of present invention encompass vaccine composition
  • vaccine composition comprising an effective immunizing amount of an isolated Salmonella spp. cell culture supernatent or CCS protein or agent, involved inType III secretion systems and a pharmaceutically acceptable carrier, wherein said vaccine composition is effective in a vertebrate subject to reduce or eliminate Gram-negative bacterial infection, or agents or compounds that modulate expression or activity of Salmonella spp. CCS gene expression to modulate or inhibit Type III secretion systems of a Salmonella spp. bacteria.
  • the agents or compounds are useful in a method for prevention or treatment of Gram negative bacterial infection such as Salmonella spp. bacterial infection.
  • An agent can be, for example, a small chemical molecule.
  • Such small chemical molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, small non-nucleic acid organic compounds or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • peptides e.g., peptoids
  • amino acids amino acid analogs
  • small non-nucleic acid organic compounds or inorganic compounds i.e., including heteroorganic and organometallic compounds having a molecular weight less than about 10,000 grams per mole
  • Exemplary doses include milligram or microgram amounts of the small chemical molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small chemical molecule depend upon the potency of the small chemical molecule with respect to the expression or activity to be modulated.
  • a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • composition aspects of the invention can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical vaccine compositions or nucleic acids, peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisory in nature and are adjusted depending on the particular therapeutic context or patient tolerance.
  • the amount of nucleic acid, peptide or polypeptide adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use i.e., the "dosing regimen” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like.
  • the mode of administration also is taken into consideration.
  • the dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest Remington's; Egleton, Peptides 18: 1431-1439, 1997; Langer, Science 249: 1527-1533, 1990.
  • compositions are administered to a patient at risk for Gram negative bacterial infection such as Salmonella spp. bacterial infection or suffering from Gram negative bacterial infection such as Salmonella spp., in an amount sufficient to at least partially arrest or prevent the condition or a disease and/or its complications.
  • Compositions are administered to a patient at risk for Gram negative bacterial carriage such as Salmonella spp. bacterial carriage or suffering from Gram negative bacterial carriage such as Salmonella spp.
  • vaccine composition comprising a soluble peptide pharmaceutical composition dosage for intravenous (IV) administration would be about 0.01 mg/hr to about 1.0 mg/hr administered over several hours (typically 1, 3, or 6 hours), which can be repeated for weeks with intermittent cycles.
  • CSF cerebrospinal fluid
  • Figure 1 shows the concentration of supernatants, then probed with a SPI-2 effector (SseB), which is a readout of the proper inducing conditions.
  • “Neat” means “not concentrated supernatants”
  • “cone” means the supernatant was concentrated (and therefore more SseB is present).
  • Supernatants were grown in either a flask or in a fermentor (at NTC).
  • Figure 1 shows that scale up for CCS production is therefore possible.
  • C57BL/6 females were used. These mice are susceptible to infection and can be used for both the streptomycin-pretreatment model of colitis and for systemic typhoid infection. A non-Sm-pretreatment model was used in this experiment and investigates colonization of the cecum, spleen and liver as endpoint measurements.
  • mice [0176] Three experimental groups of mice were used.
  • SP secreted proteins
  • MPL + TDM Emulsion from SIGMA- ALDRICH Product # M6536 Each vial is enough for 10 injections and 7 vials are required.
  • DILUENT FOR ANTIGEN AND RIBI ADJUVANT DILUENT FOR ANTIGEN AND RIBI ADJUVANT:
  • This method describes the inoculation of one mouse.
  • mice with 2 variants of the virulence proteins exhibited fewer bacteria (CFU) in the intestine, greater cecal weight (from limiting the pathological response in the cecum which usually lowers cecal weight) and less inflammation in general at several enteric sites (mucosa, epithelium, submucosa).
  • CFU bacteria
  • cecal weight from limiting the pathological response in the cecum which usually lowers cecal weight
  • inflammation in general at several enteric sites (mucosa, epithelium, submucosa).
  • each vaccine dose contained 100 ⁇ g of CCS protein while the combination CCS SPI-l/SPI-2 vaccine was formulated at 50 ⁇ g of each each antigen.
  • the placebo group which contained no antigen, included adjuvant diluted with phosphate buffered saline at the same VSA3 concentration as other groups.
  • Groups of twenty one- week-old chicks were immunized subcutaneously with each CCS formulation and were boosted 14 days later with the same vaccine. Ten days after the second immunization, all birds were challenged with 5 x 10 CFU of Salmonella Typhimurium via the oral route in a 1 ml volume.
  • Figure 1 IA shows the level of cecal colonization in each of the groups and no significant difference was observed for any of the immunized groups.
  • Figure 1 IB shows the colonization of liver and spleen and both groups which received the CCS SPI-2 vaccine showed significantly less colonization (p ⁇ .05) than any of the other groups. Colonization of chickens immunized with the CCS SPI-I formulation or the CCS SPI-l/SPI-2 formulation was not significantly different from the birds which received the placebo.

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Abstract

La présente invention concerne des procédés pour le traitement ou la prévention d'infection par des bactéries Gram négatif, telles que des espèces Salmonella, chez un sujet vertébré. Les procédés comprennent l'administration d'un antagoniste de systèmes de sécrétion de type III au sujet vertébré en une quantité efficace pour réduire ou éliminer l'infection bactérienne par une espèce Salmonella. L'invention concerne en outre des procédés pour le traitement ou la prévention d'infection par des espèces Salmonella chez des espèces aviaires ou mammifères.
PCT/CA2007/001555 2006-08-31 2007-08-31 Vaccins et procédés pour le traitement ou la prévention d'infections bactériennes par des espèces salmonella chez un sujet vertébré Ceased WO2008025171A1 (fr)

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CA002662174A CA2662174A1 (fr) 2006-08-31 2007-08-31 Vaccins et procedes pour le traitement ou la prevention d'infections bacteriennes par des especes salmonella chez un sujet vertebre

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EP2237307A2 (fr) 2009-03-30 2010-10-06 Vidal de Miguel, Guillermo Procédé et appareil pour produire des faisceaux stables d'ions sélectionnés de mobilité via des champs électriques dépendant de la durée
US20100330124A1 (en) * 2009-06-24 2010-12-30 Cowart Richard E Vaccine compositions and methods of use to protect against infectious disease
EP2432501A4 (fr) * 2009-05-22 2013-03-27 Inst Systems Biology Protéines bactériennes associées à des sécrétions pour stimuler nlrc4
WO2021055474A1 (fr) * 2019-09-16 2021-03-25 Microbiome Labs, Llc Supplémentation probiotique à base de spores chez des poulets et effet sur des charges de salmonelle
CN116254311A (zh) * 2022-12-13 2023-06-13 宁波大学 一种酶解水产动物蛋白金属螯合肽的制备方法

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2237307A2 (fr) 2009-03-30 2010-10-06 Vidal de Miguel, Guillermo Procédé et appareil pour produire des faisceaux stables d'ions sélectionnés de mobilité via des champs électriques dépendant de la durée
EP2432501A4 (fr) * 2009-05-22 2013-03-27 Inst Systems Biology Protéines bactériennes associées à des sécrétions pour stimuler nlrc4
US20100330124A1 (en) * 2009-06-24 2010-12-30 Cowart Richard E Vaccine compositions and methods of use to protect against infectious disease
US8647640B2 (en) * 2009-06-24 2014-02-11 Richard E. COWART Vaccine compositions and methods of use to protect against infectious disease
WO2021055474A1 (fr) * 2019-09-16 2021-03-25 Microbiome Labs, Llc Supplémentation probiotique à base de spores chez des poulets et effet sur des charges de salmonelle
CN116254311A (zh) * 2022-12-13 2023-06-13 宁波大学 一种酶解水产动物蛋白金属螯合肽的制备方法

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