Compositions of vaccines and adjuvants and methods for the treatment of urinary tract infections
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example 1
[0200]PHAD formulations are manufactured as follows to produce a molar ratio of about 2.5:1 of DPPC to PHAD.
[0201]PHAD, phosphorylated hexaacyl disaccharide, and DPPC can be obtained from Avanti Polar Lipids (Alabaster, Ala., USA) as either non-GMP or GMP material (the Certificate of Analysis is provided in Table 1). PHAD is a synthetic version of monophosphoryl lipid A. PHAD is formulated with DPPC to prepare the adjuvant formulation of the invention. DPPC's release specifications are provided in the table 2. The transition temperature of DPPC is 41° C.
[0202]PHAD is weighed out into an appropriate glass vial, preferably a Type 1 Plus Schott glass vial. An appropriate amount of an approximately 2.3 mg / ml DPPC solution, or equivalent, in ethanol is added. This preparation is sonicated for approximately 1 minute while gently swirling the preparation, and then the ethanol is appropriately removed via evaporation using care. The film is reconstituted with 10 mM trisodium citrate, pH 6.0...
example 2
[0207]Antigens for the vaccine may be prepared as follows.
[0208]FimCH is a non-covalent complex of FimC and FimH recombinant proteins. The recombinant proteins are derived from transgenic E. coli culture. The FimC and FimH proteins are expressed separately in E. coli, and they spontaneously form a non-covalent complex. The molecular weight of the FimCH complex is approximately 51,700 Daltons.
[0209]The FimH protein (SEQ ID No: 1) of the complex has a molecular weight of 29,065 Daltons, and it consists of 279 amino acid residues represented by the sequence below:
Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile ProIle Gly Gly Gly Ser Ala Asn Val Tyr Val Asn LeuAla Pro Val Val Asn Val Gly Gln Asn Leu Val ValAsp Leu Ser Thr Gln Ile Phe Cys His Asn Asp TyrPro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln ArgGly Ser Ala Tyr Gly Gly Val Leu Ser Asn Phe SerGly Thr Val Lys Tyr Ser Gly Ser Ser Tyr Pro PhePro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr AsnSer Arg Thr Asp Lys Pro Trp Pro Val Ala Leu TyrLeu Thr...
example 3
Bioprocessing
Process to Obtain Antigens of the Vaccine
[0214]The bioprocessing step is initiated with inoculation of master cell bank (MCB) into shake flasks containing APS LB medium with kanamycin (50 μg / mL) and chloramphenicol (20 μg / mL). When the OD reaches 2.0-3.0 units (after approximately 15 hours growth), the cell culture is transferred aseptically into reactors for fed-batch fermentation. The medium containing APS Super Broth, about 0.8% glycerol, and antibiotics is sterilized prior to inoculation. FimH protein expression is induced at OD≧10 with IPTG. Five minutes after IPTG addition, FimC protein expression is induced with arabinose. Cells are harvested approximately one hour later. After harvesting, the cells are separated from the media components by continuous or batch centrifugation.
[0216]Recombinant FimCH is expressed in the E. coli periplasm. E. coli, as a gram negative bacteria, possess an inner and an outer lipid bilayer membrane. The space bet...
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