WO2001005827A1 - Vaccins a base de fim a pilus - Google Patents

Vaccins a base de fim a pilus Download PDF

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Publication number
WO2001005827A1
WO2001005827A1 PCT/US2000/019277 US0019277W WO0105827A1 WO 2001005827 A1 WO2001005827 A1 WO 2001005827A1 US 0019277 W US0019277 W US 0019277W WO 0105827 A1 WO0105827 A1 WO 0105827A1
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Prior art keywords
adl
fima
polypeptides
polypeptide
sequence
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PCT/US2000/019277
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English (en)
Inventor
Scott J. Hultgren
Solomon Langermann
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Medimmune, Inc.
Washington University
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Publication date
Application filed by Medimmune, Inc., Washington University filed Critical Medimmune, Inc.
Priority to AU61005/00A priority Critical patent/AU6100500A/en
Priority to JP2001511484A priority patent/JP2003505044A/ja
Priority to CA002379129A priority patent/CA2379129A1/fr
Priority to EP00947385A priority patent/EP1196437A1/fr
Publication of WO2001005827A1 publication Critical patent/WO2001005827A1/fr
Priority to HK02106983.4A priority patent/HK1045701A1/zh

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    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates generally to the field of vaccines and vaccine compositions, especially those comprising pilus-proteins like FimA of Escherichia coli, wherein such proteins a present as discrete domains forming a larger polypeptide, or similar, structure.
  • bacterial infections begin with attachment of bacteria to cellular surfaces present on the host.
  • enterobacteriaceae such as Escherichia coli
  • infection begins with colonization of mucosai surfaces by the bacteria.
  • Such attachment is facilitated by the presence on the surfaces of the bacteria of structures referred to as pili, or fibrillae, or fimbriae.
  • type 1 pili which are adhesive fibers expressed in most bacteria of the Enterobacteriaceae family, facilitate the adhesive qualities of bacteria that often lead to colonization and infection of various tissues of the host animal, especially on mucosai surfaces.
  • Pili contain a short fibrillar tip structure composed of FimG, FimF and
  • FimH attached to a rod-like structure composed of FimA. More specifically,
  • FimH mediates binding to mannose-oligosaccharides present on mucosai surfaces.
  • the presence of such pili therefore plays a critical role in bacterial infection.
  • E. coli is the most common pathogen of the urinary tract, accounting for greater than 85% of cases of asymptomatic bacteriuria, acute cystitis and acute pyelonephritis, as well as greater than 60% of recurrent cystitis, and at least 35% of recurrent pyelonephritis infections. Because of the high incidence, continued persistence, and significant expense associated with E. coli urinary tract infections, there is a need for a prophylactic vaccine to reduce susceptibility to diseases such as this.
  • type 1 pili are thought to be important in initiating colonization of the bladder and inducing cystitis, whereas another type of pili, called P pili, are thought to play a role in ascending infections and the ensuing pyelonephritis.
  • Pili are heteropolymeric structures that are composed of several different structural proteins required for pilus assembly.
  • Type 1 pili-carrying bacteria recognize and bind to D-mannose in glycolipids and glycoproteins, for example, in bladder epithelial cells. Proteins forming the pili therefore make good candidates for vaccines.
  • pilus-based vaccines A major disadvantage to pilus-based vaccines has been the fact that the major immunodominant components of pilus fibers are often antigenically highly variable and therefore afford protection against only a limited number of bacterial strains.
  • pilus associated adhesins such as FimH
  • FimH are highly conserved proteins among different species and strains of bacteria. FimH is also highly conserved not only among uropathogenic strains of E. coli, but also among a wide range of gram-negative bacteria.
  • the present invention is directed to a novel class of polypeptides, and similar structures, comprising specific exposed sequences derived from the FimA molecule.
  • compositions containing the novel polypeptides disclosed according to the invention and to use such compositions to facilitate the disease treatment and prevention methods disclosed herein.
  • Figure 1 shows a comparison chart for the exposed and variable domains of FimA in diverse E. coli clinical isolates.
  • the J96 consensus sequence represents the "reference” sequence and 7 other strains are compared to it.
  • An additional strain, EC56 (not shown), has a sequence identical to J96.
  • One sequence of FimA is shown as SEQ ID NO: 24.
  • the present invention is directed to novel polypeptides formed from amino acid sequences corresponding to the different domains identified within the structure of the bacterial protein FimA, based in part on the results of X-ray structural analysis of FimH when complexed with its chaperone FimC and modeling for type 1 pilus assembly using the FimH pilin domain as a model for FimA.
  • FimA like most proteins, is composed of various "domains,” made up of amino acid sequences that form secondary structures having uniquely defined biological properties.
  • examination of a large number (over 100) of uropathogenic E. coli strains reveals the presence of a large number of subclasses of sequences within the structure of FimA (one such sequence is provided in SEQ ID NO: 24).
  • SEQ ID NO: 24 one such sequence is provided in SEQ ID NO: 24.
  • the vast majority of variations identified so far correspond to domains on the surface of the pilus organelle (based on the crystal structure of the FimCH complex and modeling for assembly).
  • the FimA gene has been isolated and sequenced from a number of strains of E. coli and the FimA sequences compared for variability. The results have indicated sequences within FimA that appear to be highly conserved and those that appear to be highly variable.
  • E. coli strains J96, EC45, NU1 4, B21 7, DS1 7, B21 2, EC42, B250 and EC56 were examined for variability in their FimA structures, wherein J96 was the reference strain.
  • the present invention is directed in part to novel polypeptides comprising one or more domains as disclosed herein. Where only one domain is present, said domain will be selected from the group consisting of the sequences of SEQ ID NO: 1 , 2, 3, 4, and 5. These sequences are conveniently referred to as domains ADL-1 , ADL-2, ADL-3, ADL-4, and ADL-5 (wherein "ADL” refers to FimA Domain Loop and the digit not only serves to distinguish one from the other but also indicates the order in which these domains occur within the FimA molecule, from N-terminal to
  • Such domains are commonly found to be present on the surface of the FimA molecule and, as such, are useful targets for antibody binding.
  • exposed sequences are readily available to form the basis for immunogenic polypeptides (especially suited for use in vaccine compositions) incorporating antigenic sites on the surface of FimA, and thus of the pilus itself. Since these are the surfaces that invading organisms show to the immune system, and since they can be readily synthesized in vitro by either recombinant or chemical synthetic means, the present invention provides easily producible immunogens for vaccination and other clinical purposes.
  • novel polypeptides of the present invention are thus purified polypeptides comprising one or more portions of FimA, each said portion independently selected from the group consisting of domains ADL-1 , ADL-2, ADL-3, ADL-4, and ADL-5 (said domains being as defined herein and not suggested as having any general or common meaning in the art), and wherein said polypeptide is other than FimA itself, regardless of the strain from which it is derived, or a polypeptide comprising FimA.
  • a purified polypeptide of the invention may contain only one such domain, in which case this would exclude any naturally occurring proteins from the disclosure herein (such as FimA itself) since none are known to include only one of these domains when in a purified state.
  • the domain could be any of the five domains just recited, with optional flanking sequences on either side of said domain so as to provide appropriate support for a more naturally occurring conformation or whatever other structural support might be needed to permit the domain sequence to attain a conformation as close to natural (and functional) as possible (such as flexibility of the molecule as a whole).
  • a polypeptide of the invention comprises more than one such domain
  • the domains are optionally attached to each other by chemical linking structures wherein said structures are of a length that is less than the length of an oligopeptide having about 20 amino acids residues (i.e., less than the length of amino acids separating any of these non-contiguous domains within a protein such as FimH).
  • the novel polypeptides of the invention would exclude FimA itself, or any protein comprising FimA.
  • such chemical linkers may be composed of amino acids or may be composed of chemical structures, such as polymers, other than amino acids or may be composed of a mixture of amino acids and other small molecules having similar spatial dimensions to amino acids. All such types of linkers are contemplated by the invention disclosed herein.
  • the preferred amino acids will be glycine and serine.
  • the latter could include homoglycine, homoserine, or glycine-serine (or gly-ser or gs) mixtures.
  • polypeptides of the present invention comprise more than one such domain, and said domains are linked by a chain of amino acids (i.e., linker amino acids), said chain will commonly be less than about 20 amino acid residues in length, preferably no more than 1 0 amino acid residues in length, and most preferably about 5 ( meaning ⁇ 1 ) residues in length.
  • linkers could be as short as 1 residue in length, or such linkers could be absent entirely and the domains linked directly to each other in a contiguous arrangement.
  • polypeptides of the present invention may contain as few as a single domain, they may also be comprised of 2, 3, or more domains, and said domains may be linked in any order.
  • the domains of the polypeptides disclosed herein will be comprised of 5 domains, most preferably ADL-1 , ADL-2, ADL-3, ADL-4, and ADL-5, and the preferred embodiment will have these domains arranged in the sequence
  • each linker would consist of a pentapeptide composed of glycine, or serine, or both, alternating or otherwise.
  • the respective linkers may be the same or may be different; if the latter, they may differ in either length or chemical identity or both.
  • one linker might be composed of only amino acids and the other might be some other type of chemical structure, such as an organic polymer.
  • polypeptide might be more loosely defined to include a structure composed of polypeptide sequences that may or may not be continuous (i.e., may or may not be themselves linked by polypeptide sequences).
  • linkers are other than common L-amino acids, synthesis of the novel polypeptides of the invention may be more complex, thus limiting the ability to prepare vaccines, and vaccine compositions, on a large-scale in a reasonably short period of time.
  • linkers are composed of amino acids, such linkers may be the same or different in sequence and may be the same or different in length.
  • orientation of the amino acid sequences within a given domain might be reversed.
  • screening might show that reversing the orientation of ADL-2 within the above sequence provides for a more antigenic structure. This could be accomplished simply by reversing the sequence of the amino acids in one or more of the domains or sequences disclosed according to the invention (by direct synthesis of the polypeptides or by synthesizing a gene to be expressed inside of a suitably engineered cell).
  • polypeptides of the invention comprise 1 , 2, 3, or more such domains. These domains may be present in any order and may include sequences in which one type of domain, for example, ADL-1 , is present in multiple copies. Similar combinations and permutations can readily be devised by those skilled in the art when 3, 4 or more such domains are linked to form a polypeptide of the present invention and all such combinations and permutations are expressly contemplated by the present invention.
  • polypeptides of the invention may have the domains with sequences oriented in an opposite direction and still be within the invention.
  • sequences oriented in an opposite direction and still be within the invention.
  • such reversals of orientation in the sequences of the amino acids might require some degree of chemical modification but all such modifications are deemed within the ordinary capabilities of those skilled in the art.
  • sequence within some or all of the ADLs of a given polypeptide, or polypeptide-like, structure are reversed while the ordering of the ADLs themselves along the polypeptide chain is the same.
  • a specific embodiment would be a polypeptide with ADLs 1 through 5 in that order (from N-terminus to C-terminus) but wherein the individual amino acid sequences within any given ADL, or some, or all, ADL's, is reversed or otherwise modified.
  • the most preferred embodiments of the present invention will be polypeptides in which the arrangement of domains will be
  • ADL-1 has the amino acid sequence of SEQ ID NO: 1
  • ADL-2 will have the amino acid sequence of SEQ ID NO: 2
  • ADL-3 has the amino acid sequence of SEQ ID NO: 3
  • ADL-4 has the amino acid sequence of SEQ ID NO: 4
  • ADL-5 has the amino acid sequence of SEQ ID NO: 5, where these sequences are the consensus sequences derived from the J96 strain as given in Figure 1
  • each linker (L) is about 5 to 10, preferably about 5, amino acids in length, including linkers with exactly 5 amino acids in length, said amino acids being all glycine, or all serine or a mixture of both, wherein said mixture may be alternating glycine and serine residues, non-alternating or may even be a random mixture.
  • linkers will no doubt suggest themselves to those of skill in the art.
  • the preferred embodiment described above would be that derived from the J96 consensus sequence depicted in Figure 1 , which sequence contains the sequences of SEQ ID NOS: 1 , 2, 3, 4, and 5.
  • the sequence of an entire FimA molecule is described by SEQ ID NO: 24 while the sequence of a preferred embodiment, with each of 5 domains appearing once, and linked by 1 0 amino acid linkers of alternating glycine and serine residues is given by SEQ ID NO: 25.
  • novel immunogenic polypeptides of the invention can also comprise functionally similar domains from other strains of E. coli, including the sequences shown in the comparison chart of Figure 1 . These sequences are also given by SEQ ID NOS: 6 through 23.
  • ADL-1 is composed of the amino acid residues numbered 32-56 of the mature FimA protein, subject to the permitted ranges and homologies described below, and include the so-called A"B loop of the FimA molecule (the AB loop in the Table below). Since the chart of Figure 1 indicates at least 5 different A"B motifs, at least 5 different peptides could be generated to cover the different possibilities.
  • a novel polypeptide and the immunogenic composition or vaccine comprising it) could include all of these ADL-1 sequences joined together as a single molecule, optionally linked by chemical linkers, preferably amino acids, most preferably made up of glycine/serine heterodimers.
  • each of the ADLs from each of the different sequences (i.e., the different strains) depicted in the chart of Figure 1 can be incorporated into a single polypeptide chain as a separate entity, each optionally linked by amino acid linkers, or otherwise joined together to form a novel and immunogenic molecular structure.
  • a novel polypeptide within the present invention could be formed by 5 different ADLs (such as ADL-1 through ADL-5) wherein each of the ADLs is derived from a different strain and thus represents a sequence joined to other sequences unlike any polypeptide sequence found in nature.
  • any of the ADLs can be present in multiple copies or not present at all within any of the novel polypeptides, or polypeptide-like structures, disclosed herein.
  • ADL-2 is a sequence derived from amino acids 60-77 of the mature FimA protein and includes the so-called BC loop. Because the chart of Figure 1 depicts at least 3 different motifs (i.e., amino acid sequences), these motifs form the basis for generation of at least 3 different peptides.
  • ADL-3 is a sequence derived from amino acids 66-92 of the mature FimA protein and includes the so-called CD' loop of FimA (CD in the Table). At least 6 different CD' motifs occur in the different strains as shown in Figure 1 and thus at least 6 different peptides can be generated from these motifs alone. These can also be joined in one contiguous molecule by using, for example, amino acid linkers of up to 20 amino acids. Novel polypeptides of the invention may thus be comprised of a series of such ADLs or any of these 6 ADLs in combination with any of the other ADLs disclosed herein.
  • ADL-4 is derived from amino acids 1 05-1 39 of the mature FimA protein, which includes the D"D loop, the D" strand, and the D"E loop of FimA (DE loop in the Table). As shown by the chart of Figure 1 , at least 6 different peptides can be formed using this motif.
  • ADL-5 is derived from amino acids 1 34-1 52 of the mature FimA protein and includes the so-called E strand and EF loop
  • domains disclosed herein are all derived from variable amino acid sequences within different strains of E. coli and all identified in the corresponding FimA molecule, it is deemed well within the ordinary skill in the biochemical arts to find amino acid replacements for one or more of the amino acids disclosed in the domain sequences of the invention so as to make them even more variable, or possibly less so. Consequently, such amino acid replacements are deemed within the present invention if said replacements number no more than about 20% of the amino acids in any of the disclosed domain sequences.
  • the present invention also encompasses any polypeptides having the disclosed domains of the invention with an arrangement, as limited by the disclosure herein, of the domains of the invention wherein said domains have amino acid sequences, individually or in combination, that are at least 80% homologous (i.e., have at least 80% sequence identity) with the domain sequences disclosed herein, and preferably at least 85% identical.
  • the invention also contemplates peptides that are at least 90% or 95% identical to the peptides of the disclosed sequences. Table 1
  • the term "percent identity” or “percent identical,” when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the described or claimed sequence (the “Reference Sequence”).
  • the Percent Identity is then determined according to the following formula:
  • C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.
  • sequences of the domains disclosed herein need not necessarily have the same lengths as said domain sequences but may be either shorter or longer.
  • longer sequences will comprise the domains disclosed herein and therefore are expressly contemplated by the present invention.
  • shorter sequences may also be contemplated by the present invention if they contain sufficiently large and functional segments or fragments of the domain sequences disclosed herein.
  • such shorter segments may comprise fragments of the domain sequences disclosed herein that contain at least 80% of the amino acids of said domain sequences.
  • ADL-1 containing the 1 5 amino acids of SEQ ID NO: 1 (for the J96 reference strain)
  • a sequence comprising any 1 3-mer (i.e., 85%) sequence within SEQ ID NO: 1 is deemed within the present invention. The same would apply to the sequences of the other domains disclosed herein.
  • portion refers to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence.
  • residues such as amino acid residues
  • fragment refers to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence.
  • the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide.
  • sequences within the invention may also be achieved by conservative substitution of the amino acids, such as by other amino acids with similar size, hydrophobicity, acidity and alkalinity.
  • substitutions need not be limited to replacement with other L-amino acids.
  • Bacterial cell wall substances are known to possess some D-amino acids and, where appropriate, the sequences disclosed herein may contain one or more such D-amino acids, or even chemically modified amino acids, if this is found to result in greater antigenicity of the novel polypeptides of the invention, provided that such substitutions follow the guidelines stated above for sequence homology and size.
  • polypeptides of the present invention may be in isolated or purified form.
  • isolated in the context of the present invention, with respect to polypeptides means that the material is removed from its original environment (e.g. , the cells used to recombinantly produce the polypeptides disclosed herein). Such peptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • the recombinant and/or synthetic immunogenic polypeptides, disclosed in accordance with the present invention, will commonly be used in a "purified” form.
  • the term “purified” does not require absolute purity; rather, it is intended as a relative definition, and can include preparations that are highly purified or preparations that are only partially purified, as those terms are understood by those of skill in the relevant art.
  • polypeptides from individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity.
  • the term "expression product” means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).
  • polypeptides of the present invention may also be present in the form of a composition.
  • Such composition where used for pharmaceutical purposes, will commonly have the polypeptide(s) of the present invention suspended in a pharmacologically acceptable diluent or excipient.
  • compositions containing one or more of the polypeptides of the invention need not be comprised of only a single kind of polypeptide.
  • immunogenic compositions can contain many different polypeptides of the invention.
  • each of the strains of FimA described in Figure 1 contains 5 different ADL domains
  • a mixture of novel polypeptides may be formed by forming a composition containing novel polypeptides containing each of the 5 domains of a particular strain where domains within a single polypeptide chain are optionally separated by linkers, the term "optionally" meaning that said linkers may or may not be present.
  • mixtures of the different ADLs may be combined to form sequences with novel combinations of the various ADLs disclosed herein and which polypeptides are then mixed together in the same compositions.
  • each of the 23 variants might be present in an immunogenic composition of the invention as a single molecule (total of 23 distinct and separate polypeptides), each ADL optionally flanked by perhaps 5 linker amino acids to give a mixture containing 23 different molecular species.
  • any combination or permutation of such species are possible.
  • a given polypeptide could contain ADL-1 from one variant strain, ADL-2 of a different variant, ADL-3 of a third variant, ADL-4 of a fourth variant and ADL-5 of a fifth variant to form a novel polypeptide of the invention containing 5 ADLs but each from a FimA of a different strain of E. coli and any number of these different species can be combined into an immunogenic composition of the invention.
  • polypeptides, or polypeptide-like structures, of the present invention can be formed where the ADL are selected from the different sequences disclosed herein.
  • these include polypeptides, and polypeptide-like structures wherein ADL-1 is selected from the group consisting of SEQ ID NO: 1 , 6, 1 0, 1 3 and 1 8, and/or wherein ADL-2 is selected from the group consisting of SEQ ID NO: 2, 7 and 9 and/or wherein ADL-3 is selected from the group consisting of SEQ ID NO: 3, 8, 1 1 , 14, 20 and 22 and/or wherein ADL-4 is selected from the group consisting of SEQ ID NO: 4, 9, 1 2, 1 5, 21 and 23, and/or wherein ADL-5 is selected from the group consisting of SEQ ID NO: 5, 1 6 and 1 7.
  • linkers utilized may also be selected for providing properties other than proper conformation or orientation.
  • linkers may be chosen for their ability to confer increased solubility properties on the polypeptide as a whole.
  • the present invention is also directed to polynucleotides capable of coding for the polypeptides of the invention, especially polynucleotides encoding the amino acid sequence of a preferred embodiment of the present invention as shown in SEQ ID NO: 25.
  • Such polynucleotides therefore contain at least one coding region for the polypeptides of the present invention, which would thus be an expression product thereof.
  • coding region refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene.
  • the coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.
  • nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
  • DNA segments encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.
  • expression product means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s) .
  • reference to a DNA sequence includes both single stranded and double stranded DNA.
  • specific sequence unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.
  • the present invention is also directed to antibodies specific for, and antisera generated in response to, polypeptides of the invention.
  • Such antibodies may be either polyclonal or monoclonal and may be generated, where monoclonal, from a cell, especially a hybridoma cell, by standard methods in the art.
  • the present invention also relates to cells, and cell lines, genetically engineered to produce such antibodies after being transfected, or otherwise transformed, so that their genomes contain, within the main chromosome or as part of a plasmid or other vector, a polynucleotide encoding the genes for an antibody specific for a polypeptide of the invention, especially where said engineered cell is a cell capable of forming and secreting a fully formed antibody, such technology being known in the art.
  • the present invention also relates to vectors, such as plasmids, comprising the polynucleotides of the invention, said polynucleotides encoding polypeptides disclosed herein, and wherein such vectors are useful for transforming cells and permitting said transformed cells to express the polypeptides of the invention.
  • the present invention also relates to cells transformed by such vectors and thereby expressing, with or without subsequent secretion thereof, of the polypeptides of the invention.
  • the present invention is also directed to vaccines containing the polypeptides disclosed herein.
  • a vaccine would comprise an immunogenically effective amount of a polypeptide of the invention.
  • a preferred embodiment of the invention is a vaccine comprising the polypeptide having the arrangement
  • each of the linkers is composed of 5 amino acid residues, and most especially where the sequence of the entire structure is the sequence of SEQ ID NO: 25 (which is composed of the J96 strain ADL sequences of Figure 1 ).
  • a vaccine or to produce antibodies for use as a diagnostic or as a passive vaccine
  • proteins and fragments are contemplated.
  • the invention disclosed herein relates to an immunogenic composition
  • an immunogenic composition comprising a purified polypeptide, said polypeptide comprising a portion of FimA, said portion selected from the group consisting of ADL-1 , ADL-2, ADL-3, ADL-4, and ADL-5, wherein said polypeptide is other than FimA or a polypeptide comprising FimA.
  • the purified polypeptides comprising the immunogenic compositions of the present invention do not include FimA itself, or pre-FimA, or any other polypeptide or protein comprising FimA or its preprotein.
  • the portion of FimA contained in said polypeptides may be derived from a highly conserved region of FimA or from a highly variable region of FimA, as those terms are understood in the art, and wherein said FimA is the FimA found in any of the bacteria of the enterobacteriaceae family, such as E. coli.
  • an immunogenic composition according to the invention may be utilized to produce antibodies to diagnose urinary tract infections, or to produce vaccines for prophylaxis and/or treatment of such infections as well as booster vaccines to maintain a high titer of antibodies against the immunogen(s) of the immunogenic composition.
  • antibodies may be generated using the polypeptides of the present invention for research purposes, as a means of studying protein-antibody binding and interactions.
  • vaccines are prepared as injectables, in the form of aqueous solutions or suspensions. Vaccines in an oil base are also well known such as for inhaling. Solid forms which are dissolved or suspended prior to use may also be formulated.
  • Pharmaceutical carriers are generally added that are compatible with the active ingredients and acceptable for pharmaceutical use. Examples of such carriers include, but are not limited to, water, saline solutions, dextrose, or glycerol. Combinations of carriers may also be used.
  • the pharmaceutical compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • a pharmaceutically acceptable carrier including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Vaccine compositions may further incorporate additional substances to stabilize pH, or to function as adjuvants, wetting agents, or emulsifying agents, which can serve to improve the effectiveness of the vaccine.
  • Vaccines are generally formulated for parenteral administration and are injected either subcutaneously or intramuscularly. Such vaccines can also be formulated as suppositories or for oral administration, using methods known in the art.
  • the amount of vaccine sufficient to confer immunity to pathogenic bacteria is determined by methods well known to those skilled in the art. This quantity will be determined based upon the characteristics of the vaccine recipient and the level of immunity required. Typically, the amount of vaccine to be administered will be determined based upon the judgment of a skilled physician. Where vaccines are administered by subcutaneous or intramuscular injection, a range of 50 to 500 ⁇ g purified protein may be given.
  • polypeptides of the present invention can be used as immunogens to stimulate the production of antibodies for use in passive immunotherapy, for use as diagnostic reagents, and for use as reagents in other processes such as affinity chromatography.
  • Recombinant polypeptides of the invention will commonly result from the engineering of the amino acid sequence of the domains disclosed herein with appropriate linker structures to provide for conformational flexibility and to meet stereospecific needs in generating appropriate structures for use as immunogens. This is readily accomplished by engineering the appropriate DNA sequence, inserting this sequence into a vector and then transforming the appropriate cells to express the desired polypeptides. Such an approach may then be used to produce a cell line that stably expresses the genetically engineered polypeptide.
  • polypeptides of the present invention can be readily synthesized by chemical means, especially automated means, well known in the biochemical art.
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a non-human. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used.
  • Examples include the hybridoma technique (Kohler and Milstein, 1 975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1 983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1 985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
  • cells can be transformed with gene sequences corresponding to antibody chains containing variable regions complementary to the polypeptides of the invention and thereby generate engineered antibodies to the polypeptides disclosed herein.
  • the present invention is also directed to the uses of the disclosed polypeptides as vaccines to treat diseases caused by bacterial species and to the uses of antibodies specific for the polypeptides of the invention in treating such diseases.
  • the present invention is directed to a method of preventing a disease in an animal at risk thereof comprising administering to said animal the vaccines according to the invention, especially where said disease is a disease caused by a bacterium of the family enterobacteriaceae, most especially when the bacterium is E. coli and most preferably where the animal is a human.
  • the present invention is also directed to a method of treating a disease in an animal afflicted therewith comprising administering to said animal a pharmacologically effective amount of the composition of the antibodies specific for the polypeptides disclosed herein, wherein said antibodies are suspended in a pharmacologically acceptable diluent or excipient and are present in a sufficient amount to result in amelioration of the disease condition, especially where the bacterium is of the family enterobacteriaceae and most especially where the bacterium is E. coli, and most preferably where the animal to be treated is a human.

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Abstract

L'invention concerne de nouveaux polypeptides formés à partir de séquences aminoacides correspondant à des domaines exposés sur la surface de Fim A, protéine constituant une zone de pili de type de bactéries telles que E. coli. Elle traite en outre de compositions contenant ces nouveaux polypeptides à utiliser comme vaccins et servant à produire des anticorps spécifiques à utiliser dans le traitement et/ou la prévention de maladies, telles que des infections des voies urinaires, provoquées par des bactéries de la famille des enterobacteriaceae.
PCT/US2000/019277 1999-07-15 2000-07-14 Vaccins a base de fim a pilus WO2001005827A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU61005/00A AU6100500A (en) 1999-07-15 2000-07-14 Fima pilus-based vaccines
JP2001511484A JP2003505044A (ja) 1999-07-15 2000-07-14 ＦｉｍＡピリ線毛ベイストワクチン
CA002379129A CA2379129A1 (fr) 1999-07-15 2000-07-14 Vaccins a base de fim a pilus
EP00947385A EP1196437A1 (fr) 1999-07-15 2000-07-14 Vaccins a base de fim a pilus
HK02106983.4A HK1045701A1 (zh) 1999-07-15 2002-09-25 Fima菌毛基疫苗

Applications Claiming Priority (2)

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US14401399P 1999-07-15 1999-07-15
US60/144,013 1999-07-15

Publications (1)

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WO2001005827A1 true WO2001005827A1 (fr) 2001-01-25

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EP (1) EP1196437A1 (fr)
JP (1) JP2003505044A (fr)
AU (1) AU6100500A (fr)
CA (1) CA2379129A1 (fr)
HK (1) HK1045701A1 (fr)
WO (1) WO2001005827A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5417986A (en) * 1984-03-16 1995-05-23 The United States Of America As Represented By The Secretary Of The Army Vaccines against diseases caused by enteropathogenic organisms using antigens encapsulated within biodegradable-biocompatible microspheres
WO1998005348A1 (fr) * 1996-08-02 1998-02-12 Department Of The Army, U.S. Government Peptides sensibles a des anticorps diriges contre un peptide consensus des proteines de la famille c54-cfa/i
US5914114A (en) * 1995-06-02 1999-06-22 The United States Of America As Represented By The Secretary Of The Army Method of raising antibodies against E. coli of the family CS4-CFA/I

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5417986A (en) * 1984-03-16 1995-05-23 The United States Of America As Represented By The Secretary Of The Army Vaccines against diseases caused by enteropathogenic organisms using antigens encapsulated within biodegradable-biocompatible microspheres
US5914114A (en) * 1995-06-02 1999-06-22 The United States Of America As Represented By The Secretary Of The Army Method of raising antibodies against E. coli of the family CS4-CFA/I
WO1998005348A1 (fr) * 1996-08-02 1998-02-12 Department Of The Army, U.S. Government Peptides sensibles a des anticorps diriges contre un peptide consensus des proteines de la famille c54-cfa/i

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
THANKAVEL KRISHNAN ET AL: "Localization of a domain in the FimH adhesion of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection.", JOURNAL OF CLINICAL INVESTIGATION, vol. 100, no. 5, September 1997 (1997-09-01), pages 1123 - 1136, XP002156010, ISSN: 0021-9738 *

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AU6100500A (en) 2001-02-05
EP1196437A1 (fr) 2002-04-17
CA2379129A1 (fr) 2001-01-25
JP2003505044A (ja) 2003-02-12

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