WO2001005827A1

WO2001005827A1 - Fima PILUS-BASED VACCINES

Info

Publication number: WO2001005827A1
Application number: PCT/US2000/019277
Authority: WO
Inventors: Scott J. Hultgren; Solomon Langermann
Original assignee: Medimmune, Inc.; Washington University
Priority date: 1999-07-15
Filing date: 2000-07-14
Publication date: 2001-01-25
Also published as: CA2379129A1; EP1196437A1; AU6100500A; HK1045701A1; JP2003505044A

Abstract

Novel polypeptides formed from amino acid sequences corresponding to exposed domains on the surface of FimA, a protein making up a portion of type 1 pili of bacteria, such as E. coli, are disclosed. Also disclosed are compositions containing these novel polypeptides for use as vaccines and for generating specific antibodies for use in treatment and/or prevention of diseases, such as urinary tract infections, caused by bacteria of the family enterobacteriaceae.

Description

FimA PILUS-BASED VACCINES

This application claims priority of U.S. Provisional Application 60/1 44,01 3, filed 1 5 July 1 999, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Many kinds of bacterial infections begin with attachment of bacteria to cellular surfaces present on the host. For example, in bacterial infections caused by 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. For example, in gram negative bacteria, such as E. coli, 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. Such adhesion to epithelial cell surfaces is facilitated by the presence in the pilus of a protein called an "adhesin," of which FimH is an example. 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.

Because colonization of mucosai epithelium, such as in the urinary tract, is widely accepted as a prerequisite to infection, the disruption or prevention of pilus-mediated attachment of bacteria, such as E. coli, to urinary epithelia is expected to prevent or retard the development of urinary tract infections.

For example, 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.

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. Conversely, pilus associated adhesins, such as 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.

Thus, attempts at generating pilus vaccines based on whole pili have often failed due to the antigenic variability, from one isolate to another, of the proteins making up the pilus. For example, studies with whole type 1 pili have demonstrated about a 50% serological heterogeneity [See: Guerina et al. Infect. Immun. 57: 1 568-1 572 (May, 1 989)] although nothing was known about specific sequences of proteins, such as FimA, among these strains. Still, attempts have been made to provide practical applications to the use of whole type 1 pili [U.S. Pat. No. 4,454, 1 1 7].

Despite the poor results using vaccines based on whole pili, use of specific proteins present within the pili, either having highly conserved segments or having variable segments that might still be exploited for clinical purposes, offers an opportunity to provide methods for combating diseases caused by gram-negative organisms, such as E. coli.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a novel class of polypeptides, and similar structures, comprising specific exposed sequences derived from the FimA molecule.

It is an object of the present invention to provide amino acid sequences corresponding to identified domains of pilins like FimA, such domains being derived from studies on the structures of different E. coli isolates.

It is also an object of the present invention to provide novel polypeptides containing amino acid sequences corresponding to highly variable regions, or portions, or domains of FimA, derived from different isolates of E. coli, and arranged so as to form a single polypeptide structure, which structure elicits an immunogenic response when injected into an animal, such as a human.

It is a further object of the present invention to provide genetically engineered or chemically synthesized polypeptides containing at least two or more of the sequences corresponding to the domains identified according to the invention and to provide methods of using such polypeptides as immunogens for eliciting antibodies.

It is also an object of the invention to provide immunogenic compositions containing purified polypeptides, other than FimA itself, or any polypeptides containing FimA, wherein such polypeptides comprise selected immunogenic portions of the FimA molecule, especially where such portions include the variable loops disclosed herein.

It is a still further object of the present invention to provide nucleotide sequences coding for such engineered polypeptides.

It is yet a further object of the present invention to provide novel polypeptides useful as vaccines for prevention and/or treatment of diseases caused by bacteria of the family enterobacteriaceae, as well as methods for using compositions of such vaccines (i.e., vaccine compositions) to treat and/or prevent such diseases, especially in humans.

It is another object of the present invention to provide for antibodies specific for the polypeptides, and polypeptide-like structures, disclosed herein for use in treating diseases caused by bacteria from which the adhesin structure was derived, especially E. coli.

It is yet a still further object of the present invention to provide for 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.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows a comparison chart for the exposed and variable domains of FimA in diverse E. coli clinical isolates. Here, 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.

DETAILED DESCRIPTION OF THE INVENTION

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. In accordance with the invention disclosed herein, 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). The vast majority of variations identified so far (based on sequence analysis) correspond to domains on the surface of the pilus organelle (based on the crystal structure of the FimCH complex and modeling for assembly). [Choudhury et al, X-ray Structure of the FimC-FimH Chaperone-Adhesin Complex from Uropathogenic E. coli, Science 285, 1 061 (1 999); Sauer et al, Structural Basis of Chaperone Function and Pilus Biogenesis, Science 285, 1058 (1 999); Barnhart et al., PapD-like Chaperones Provide the Missing Information for Folding of Pilin Proteins, Proc. Natl. Acad. Sci. USA, 10,1 073/pnas.1 301 83897 (published online June 20, 2000), the disclosures of which references are hereby incorporated by reference in their entirety].

In addition, analyses performed on sequential E. coli isolates from women with recurrent urinary tract infections have demonstrated that the same E. coli may be responsible for recurrent infections and that, among the strains responsible for recurrent infections, the FimA sequences are highly conserved from episode to episode. Consequently, there does not appear to be selective pressure during natural infections toward hypervariability of the FimA protein itself within the same individual.

In accordance with the present invention, 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.

It is therefore an object of the present invention to use the results of such analyses as a means of generating vaccines, and vaccine compositions, comprised of immunodominant regions of FimA from major classes of FimA identified using diverse E. coli clinical isolates. Such vaccines can be used alone or in conjunction with other vaccines, such as those based on FimH, and will find use against invasive E. coli infections, for example, sepsis caused by E. coli K1 strains, or more uropathogenic E. coli strains. In accordance with the present invention, 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.

Generation of the amino acid sequences (plus flanking regions) to form the peptides critical to binding is rendered a formality by sequence information disclosed herein, thereby providing for the formation of more specific, highly potent pilus-based vaccine compositions. Thus, antigenic molecules with either highly conserved or variable regions are readily available for screening as vaccines.

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

C-terminal direction).

Such domains are commonly found to be present on the surface of the FimA molecule and, as such, are useful targets for antibody binding. Thus, such 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. In addition, because the structures are limited in size and can be readily prepared, a means is provided for previously difficult to achieve large-scale production of useful vaccines, and vaccine compositions, for preventing and/or treating many diseases involving bacteria of the enterobacterial variety, especially uropathogenic organisms.

The 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. In such case, 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).

Where 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). Thus, the novel polypeptides of the invention would exclude FimA itself, or any protein comprising FimA. In addition, 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. Where said chemical linkers are composed of amino acids, the preferred amino acids will be glycine and serine. The latter could include homoglycine, homoserine, or glycine-serine (or gly-ser or gs) mixtures.

When 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. Of course, such 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.

While the 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.

Most preferably, 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

NH₂-(ADL-D— L— (ADL-2)— L— (ADL-3)— L— (ADL-4)— L— (ADL-δ)-COOH

wherein the domains form an unbroken amino acid sequence from the N- terminal end of ADL-1 to the C-terminal end of ADL-5, where "L" denotes a linker (preferably formed of amino acids) and wherein the size of the linkers, or the presence of linkers at all, is optional. In the most preferred embodiment, each linker would consist of a pentapeptide composed of glycine, or serine, or both, alternating or otherwise. In addition, 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. Thus, 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. In the latter case, the term "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). Of course, where the 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. In addition, where the 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.

Of course, it is also contemplated by the disclosure herein that the orientation of the amino acid sequences within a given domain might be reversed. For example, 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).

It should be reiterated that the 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.

In addition, as stated, the polypeptides of the invention may have the domains with sequences oriented in an opposite direction and still be within the invention. Of course, 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. In one such embodiment, the 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.

As stated, the most preferred embodiments of the present invention will be polypeptides in which the arrangement of domains will be

wherein 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, and ADL-3 has the amino acid sequence of SEQ ID NO: 3, ADL-4 has the amino acid sequence of SEQ ID NO: 4, and 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 , and wherein 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. Other linkers will no doubt suggest themselves to those of skill in the art.

Of course, 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.

The 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.

More particularly, 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. Of course, 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. In addition, said ADL-1 domains, such as the ADL-1 of EC45 (SEQ ID NO: 6) can be linked to the other kinds of domains from J96, or any of the other strains, to form a totally novel arrangement. Thus, the invention disclosed herein expressly contemplates that for the purpose of assembling a novel polypeptide within the invention, 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.

By way of non-limiting example, 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. In addition, 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.

Also in keeping with the present invention, 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.

In addition, ADL-3, as disclosed herein, 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.

In the same way, 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.

In the same way, 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

(EF loop in the Table). As shown by the chart of Figure 1 , at least 3 different peptides can be generated from this sequence and can optionally be joined with linkers.

Available ADL sequences disclosed according to the invention are summarized in Table 1 wherein the sequences are also to be found in Figure 1 .

In addition, it should be kept in mind that while the 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.

In addition, 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

SEQ ID NO: SEQUENCE STRAIN LOOP

1 GQVRTASLAQEGATS J96 AB

2 TNVASKAA BC

3 DAGHTNV CD

4 GAALTLDGATFSSETTLNNGTNTIP DE

5 YFAGAATP EF

6 GQVRTATLKQAGATS EC45 AB

7 TTVATKAA BC

8 DSTHPKV CD

9 GNELTLDGATFSSETTLNNGTNTIP DE

10 GQVRTASLKQAGATS B21 7 AB

1 1 DATHTDV CD

1 2 GAALALDGATFSSETTLNNGTNTIP DE

1 3 GQVRTASLAQDGATS DS1 7 AB

14 DTNHTNV CD

1 5 GKTLALDGASFSAQTTLNNGTNTIP DE

1 6 YYAGIATP EF

1 7 YFAGIATP B21 2 EF

1 8 GQVRTASLKQTGATS EC42 AB

1 9 TSVATKAA BC

20 DSAHPKV CD

21 GNELTLDGATFSAQTTLNNGTNTIP DE

22 DATHRDV B250 CD

23 GNALTLDGATFSAQTTLNNGTNTIP DE

In accordance with the present invention, 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:

Percent Identity = 100 [1 -(C/R)]

wherein 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.

If an alignment exists between the Compared Sequence and the

Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified Percent Identity.

In addition, the sequences of the domains disclosed herein, in order to be contemplated by the present invention, need not necessarily have the same lengths as said domain sequences but may be either shorter or longer. Of course, longer sequences will comprise the domains disclosed herein and therefore are expressly contemplated by the present invention. Conversely, 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. Thus, 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.

By way of non-limiting example, for 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.

As used herein, the terms "portion," "segment," and "fragment," refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were subjected to treatment with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide.

Different 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. In addition, such 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.

It is also contemplated that the polypeptides of the present invention may be in isolated or purified form. The term "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. For example, polypeptides from individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity.

For purposes of recombinantly producing the polypeptides of the invention, 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).

Thus, the 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.

Of course, compositions containing one or more of the polypeptides of the invention need not be comprised of only a single kind of polypeptide. Thus, it is certainly contemplated by the disclosure herein that such immunogenic compositions can contain many different polypeptides of the invention. For example, while each of the strains of FimA described in Figure 1 contains 5 different ADL domains, it is expressly contemplated by the invention as disclosed herein that 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. In addition, 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.

It is even possible to form a single molecule containing all 23 different domain loop variants (shown in Figure 1 ), all joined by flexible gly/ser linkers. Alternatively, 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. Thus, any combination or permutation of such species are possible.

In addition, 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.

In specific embodiments, the polypeptides, or polypeptide-like structures, of the present invention can be formed where the ADL are selected from the different sequences disclosed herein. In separate embodiments, 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.

In addition, regardless of the types of domains being linked to form the polypeptides of the present invention, the linkers utilized may also be selected for providing properties other than proper conformation or orientation. For example, such 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.

As used herein, the term "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.

In accordance with the present invention, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. Generally,

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.

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) .

As used herein, reference to a DNA sequence includes both single stranded and double stranded DNA. Thus, the 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. In addition, 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. Such 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

wherein 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 ).

It is an object of the present invention to utilize as immunogenic composition for a vaccine (or to produce antibodies for use as a diagnostic or as a passive vaccine) comprising a novel fusion polypeptide of the invention. In one embodiment, proteins and fragments (naturally or recombinantly produced, as well as functional analogs) of these polypeptides are contemplated.

In one embodiment, the invention disclosed herein relates to 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. Thus, 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. For the polypeptides that form the immunogenic compositions of the invention, 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.

In another aspect of the invention, 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.

In addition, such antibodies may be generated using the polypeptides of the present invention for research purposes, as a means of studying protein-antibody binding and interactions.

While other antigens have been utilized to produce antibodies for diagnosis and for the prophylaxis and/or treatment of bacterial urinary tract infections, there is a need for improved or more efficient vaccines. Such vaccines should have an improved or enhanced effect in preventing bacterial infections mediated by adhesins and pili.

Generally, 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 thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ. current edition).

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 μg purified protein may be given.

In addition to use as vaccines, the polypeptides of the present invention, and immunogenic fragments thereof, 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.

Of course, such recombinant expression cannot be used when the chemical linkers are of a chemical nature other than that of simple chains of amino acids.

In addition, because the amino acid sequences disclosed herein are not very long, even when as many as 5 domains are used and linkers are as much as 20 amino acids in length, the polypeptides of the present invention can be readily synthesized by chemical means, especially automated means, well known in the biochemical art.

The 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).

Techniques described for the production of single chain antibodies

(U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention. In addition, 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.

Thus, 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.

Claims

WHAT IS CLAIMED IS:

1 . An isolated polypeptide comprising one or more portions of a FimA, each said portion independently selected from the group consisting of domains ADL-1 , ADL-2, ADL-3, ADL-4, and ADL-5, wherein said polypeptide is other than FimA or a polypeptide comprising FimA.

2. The isolated polypeptide of claim 1 , wherein the domains are linked together by linker amino acid chains different from those found in FimA.

3. The isolated polypeptide of claim 2, wherein the linker amino acid chains are no greater than 20 amino acids in length.

4. The isolated polypeptide of claim 3, wherein the linker amino acid chains are each 5 amino acids in length.

5. The isolated polypeptide of claim 2, wherein the linker amino acid chains are composed of amino acids selected from the group consisting of glycine and serine.

6. The isolated polypeptide of claim 1 , wherein said polypeptide contains at least 5 portions of FimA selected from the group consisting of ADL- 1 , ADL-2, ADL-3, ADL-4, and ADL-5.

7. The isolated polypeptide of claim 6, wherein said polypeptide contains the portions of FimA in the order ADL-1 , ADL-2, ADL-3, ADL-4, and ADL-5 from N-terminal to C-terminal.

8. The isolated polypeptide of claim 7, wherein the portions are linked to each other by linker amino acid sequences consisting of 5 amino acids selected from the group consisting of glycine and serine.

20. A method of preventing a disease in an animal at risk thereof comprising administering to said animal the vaccine composition of claim 1 9.

21 . The method of claim 20 wherein the disease is caused by a bacterium of the family enterobacteriaceae.

22. The method of claim 20 wherein the bacterium is E. coli.

23. The method of claim 20 wherein the animal is a human.

24. A method of treating a disease in an animal afflicted therewith comprising administering to said animal a pharmacologically effective amount of the composition of claim 1 8.

25. The method of claim 24 wherein the disease is caused by a bacterium of the family enterobacteriaceae.

26. The method of claim 25 wherein the bacterium is E. coli.

27. The method of claim 24 wherein the animal is a human.

28. The method of claim 27 wherein the disease is a urinary tract infection.

29. The method according to claim 28 wherein the urinary tract infection is caused by E. coli.