Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated

Definitions

• multocida genes that have been demonstrated to exist in other species (e.g. P. (Mannheimia) haemolytica. A. pleuropneumoniae and H. somnus) include genes exbB, atpG, pnp, guaB and yjgF.
• Attenuated P. multocida strains identified using STM are insertional mutants wherein a virulence gene has been rendered non-functional through insertion of transposon sequences in either the open reading frame or regulatory DNA sequences. These insertional mutants still contain all of the genetic information required for bacterial virulence and can possibly revert to a pathogenic state by deletion of the inserted transposon.
• a strategy using counterselectable markers can be employed which has commonly been utilized to delete genes in many bacteria.
• a double selection strategy is often employed wherein a plasmid is constructed encoding both a selectable and counterselectable marker, with flanking DNA sequences derived from both sides of the desired deletion.
• the selectable marker is used to select for bacteria in which the plasmid has integrated into the genome in the appropriate location and manner.
• the counterselecteable marker is used to select for the very small percentage of bacteria that have spontaneously eliminated the integrated plasmid.
• Proteins may be produced directly or fused to a peptide or polypeptide, and either intracellularly or extracellularly by secretion into the periplasmic space of a bacterial cell or into the cell culture medium.
• Secretion of a protein requires a signal peptide (also known as pre-sequence); a number of signal sequences from prokaryotes and eukaryotes are known to function for the secretion of recombinant proteins.
• the signal peptide is removed by signal peptidase to yield the mature protein.
• the virulence gene products produced by the methods described above are used in high throughput assays to screen for inhibitory agents.
• the sources for potential agents to be screened are chemical compound libraries, fermentation media of Streptomycetes, other bacteria and fungi, and cell extracts of plants and other vegetations.
• assays are established based on the activity, and a large number of potential agents are screened for ability to inhibit the activity.
• binding assays are established to measure such interaction directly, and the potential agents are screened for ability to inhibit the binding interaction.
• the use of different assays known in the art is contemplated according to this aspect of the invention.
• Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol 5:701-707 (1997).
• the two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA-binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.
• UAS upstream activation sequence
• the assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.
• each plate was pooled to form the "input pool" by combining 100 ⁇ l from each of the wells of the micro-titer plate.
• the culture was diluted appropriately in BHI to doses of approximately 10", 10 5 , 10 6 CFU/ml and 0.2 ml of each dilution was used to infect female 14-16 g BALB/c mice by intraperitoneal administration.
• one or two surviving mice were euthanized and the spleens harvested. The entire spleen was homogenized in 1.0 ml sterile 0.9 % saline.
• the clones and their approximate LD 50 values are listed in Table 1.
• a control experiment with the wild type strain was run in parallel with each set of challenges and in all cases mortality in wild type-challenged groups was 100%.
• flanking sequence was determined for each of the 110 mutants by inverse PCR and direct product sequencing. Inverse PCR was used to generate flanking DNA products for direct sequencing as described above.
• Example 9 The putative identities listed in Table 3 (below, Example 9) were assigned by comparison with bacterial databases.
• the 110 mutants represented 35 groups of unique transposon insertions. The number of different mutations per loci varied, with some clones always containing an insertion at a single site within an ORF to clones containing insertions within different sites of the same ORF. Three multiple insertions were detected in the 110 mutants screened as determined by production of multiple PCR bands and generation of multiple sequence electropherograms.
• Example 9 The putative identities listed in Table 3 (below, Example 9) were assigned by comparison with bacterial databases.
• the 110 mutants represented 35 groups of unique transposon insertions. The number of different mutations per loci varied, with some clones always containing an insertion at a single site within an ORF to clones containing insertions within different sites of the same ORF. Three multiple insertions were detected in the 110 mutants screened as determined by production of multiple PCR bands and generation of multiple sequence electropherogram
• Legionella pneumophila homolog, mip [Engleberg, et al, Infect Immun. 57:1263- 1270 (1989)], is responsible for virulence and infection of macrophages [Cianciotto, et al, J. Infect. Dis. 762:121-6 (1990); Cianciotto, et al, Infect. Immun. 57:1255- 1262 (1989)].
• Tig, or trigger factor [Crooke and Wickner, Proc. Natl. Acad. Sci. USA. 54:5216-20 (1987), Guthrie, and Wickner, J Bacteriol. 172:5555-62 (1990), reviewed in Hesterkamp, and Bukau., FEBS Lett.
• a dnaK mutant of Vibrio cholera affected the production of ToxR and its regulated virulence factors in vitro but similar results were not obtained from in vivo grown cells [Chakrabarti, et al. Infect Immun. 67:1025-1033 (1999)].
• the CI of A. pleuropneumonia dnaK mutant was higher than most of the attenuated mutants although still approximately half of the positive control strain.

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