WO2001040280A2 - Composition and method for treating a microbial infection - Google Patents

Abstract

The invention provides polypeptides derived from regions of flagellin polypeptides than can be used to generate an immune response to gram-negative bacteria. Also provided are antibodies to the flagellin polypeptides and methods of treating a gram negative bacterial infection using the polypeptides or the antibodies.

Description

COMPOSITION AND METHOD FOR TREATING A MlCROBIAL

INFECTION

FIELD OF THE INVENTION

The present invention relates to generally to compositions and methods for treating infections caused by gram negative bacteria and more specifically to methods of treating infections caused by gram negative bacteria using antibodies to fragments of a flagellin polypeptide.

BACKGROUND OF THE INVENTION

Flagellin is a monomeric sub-unit of bacterial flagella, a polymeric rod-like appendage extending from the outer membrane of gram-negative bacteria that propels the organism through its aqueous environment. Unlike the eukaryotic flagellum, which is relatively large and contains many proteins, the bacterial filament is much smaller in size and composed of several thousand copies of a monomeric protein, flagellin. The M_r of flagellin varies greatly among bacteria, ranging from 28,000 to 60,000. Despite this variation in M_r, nucleic acid and peptide analyses of flagellin from many genera show considerable homology and sequence conservation.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery that antibodies raised in a host to amino terminal regions of the flagellin polypeptide confer resistance in the host to infections by gram negative bacteria. Accordingly, in one aspect, the invention features a polypeptide fragment containing the amino terminal region of a flagellin polypeptide or the carboxy terminal region of a flagellin polypeptide. Preferably, the polypeptide fragment includes an epitope that binds to an antibody that neutralizes a gram-negative bacterial infection. Also within the invention is a fusion polypeptide that includes the amino terminus of a flagellin polypeptide.

In a further aspect, the invention provides a nucleic acid encoding a fragment of a flagellin polypeptide or a nucleic acid encoding a fusion polypeptide containing at least a fragment of a flagellin polypeptide. The invention also features a vector containing a nucleic acid encoding a flagellin polypeptide fragment or flagellin polypeptide fusion polypeptide. Also within the invention is a cell containing a nucleic acid encoding a flagellin polypeptide fragment or a fusion polypeptide, e.g., a vector containing a nucleic acid encoding a flagellin polypeptide fragment.

Also provided by the invention is an antibody that binds specifically to a flagellin polypeptide fragment. The antibody can be, e.g., a polyclonal antibody or a monoclonal antibody

In another aspect, the invention provides a pharmaceutical composition that includes a flagellin polypeptide fragment (e.g., an amino terminal or carboxy terminal fragment of a flagellin polypeptide), or a nucleic acid encoding flagellin polypeptide fragment (e.g., an amino terminal or carboxy terminal fragment of a flagellin polypeptide). Also within the invention is a pharmaceutical composition that includes an antibody that binds specifically to a flagellin polypeptide fragment.

In a still further aspect, the invention provides a method of inhibiting an infection associated with a gram negative bacterium in a subject by administering to the subject a protein that includes a fragment of a flagellin polypeptide, a nucleic acid encoding the protein, or an antibody to a protein that includes a flagellin polypeptide. The subject can be, e.g., a mammal such as a human, horse, cow, dog, cat, mouse, rat, pig, goat, sheep, and rabbit. The method can be used prophylactically, i.e., to prevent an infection associated with a gram-negative bacteria, or therapeutically, i.e., to treat an infection associated with a gram negative bacteria.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are representations of the results of blot analyses of flagellin proteins probed with anti-sera raised against S. muenchen flagellin protein.

FIG. 2 is a histogram showing the effect of lapine ant-flagellin antiserum on the infectivity of a pathogenic E. coli in a murine model of urinary tract infection.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for producing broad-spectrum active and passive humoral immunity to flagellin polypeptides from gram negative bacteria. The composition can be used to prevent and/or treat a broad spectrum of gram negative infections in a subject, such as a human.

The invention is based in part on the discovery that incubation of a pathogenic E. coli with antisera raised against the amino terminal region of a Salmonella muenchen flagellin reduces the infectivity of the pathogenic E. coli. Moreover, passive immunization of a mice with antiserum raised against the amino terminal region of Salmonella muenchen flagellin, as well as immunization of mice with the polypeptide, is protective against subsequent challenge by gram-negative bacteria.

Accordingly, the invention includes a polypeptide containing the amino terminal sequence of a flagellin polypeptide. The polypeptide can be introduced into a subject and used to inhibit, reduce, or prevent infections associated with gram negative bacteria.

Also provided are antibodies raised against a polypeptide containing the amino terminal sequence of a flagellin polypeptide.

Polypeptides containing the amino terminal sequence or carboxy sequence of a flagellin polypeptide

A preferred flagellin polypeptide includes a polypeptide sequence derived from the amino terminal region or carboxy terminal region of a bacterial flagellin polypeptide. Preferably, the polypeptide includes a sequence highly conserved between two or more gram negative bacterial species. An example of a suitable polypeptide sequence is shown in Table 1 : Shown is the amino acid sequence of the amino-terminal region (amino acids 1-156) of Salmonella muenchen flagellin :

Table 1

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSG RINSAKDDAAGQAIANRFTA IKGLT QASRNANDGISIAQTTEGALNEI KNLQRVRELAVQSANGTNSQSDLDSIQAEITQRLNEID RVSGQTQFNGVKVLAQDNTLTIQVGANDGETI (SEQ ID NO:l)

The amino terminal regions of flagellin polypeptides of other bacteria can also be used. Other suitable bacteria include, e.g., Salmonella spp., an Aeromonas spp., a Yersinia spp., a Proteus spp., a Serratia spp. a Pseudomonas spp. and a Vibrio spp.

In a preferred embodiment, the flagellin polypeptide fragment includes at least one epitope that binds to an anti-flagellin antibody. Preferably, the antibody is neutralizing a gram-negative bacterial infection in a subject.

In some embodiments, the polypeptide varies in sequence from that of the amino terminal region of a naturally occurring flagellin polypeptide. For example, in preferred embodiments the sequence is at least 85%o identical to the corresponding amino acid sequence of amino acids 1 to 156 oϊ & Salmonella muenchen flagellin polypeptide, e.g. , a polypeptide that includes the amino acid sequence of SEQ ID NO:l. More preferably, the polypeptide is at least 90%, 92%, 95%, 97%, 98%, or 99% or more identical to the amino acid sequence of a Salmonella muenchen polypeptide (e.g., the polypeptide sequence shown in SEQ ID NO:l). For example, the variant polypeptide may include one or more conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO:l.

The phrases "percent identity" or "% identity" refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc., Madison Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homo logy between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the method described in Hein, J. (1990) Methods Enzynol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.

In addition to naturally-occurring variants of a flagellin polypeptide sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence encoding a flagellin polypeptide (e.g., the sequence of SEQ ID NO:2), thereby leading to changes in the amino acid sequence of the encoded flagellin protein, without altering the ability of the flagellin polypeptide fragment to elicit a neutralizing immune response. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:2. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a flagellin polypeptide without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the flagellin polypeptide or polypeptide fragments of the present invention are predicted to be particularly unamenable to alteration.

Preferred variants are those that have conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a flagellin polypeptide is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a flagellin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for the neturalizing antibody-inducing activity to identify mutants that retain activity.

A flagellin polypeptide, or fragment of a flagellin polypeptide, is preferably provided as a purified polypeptide. By "purified polypeptide" is meant a polypeptide or biologically active portion thereof that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the flagellin polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of flagellin polypeptide in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of flagellin polypeptide having less than about 30% (by dry weight) of non-flagellin polypeptide (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-flagellin polypeptide, still more preferably less than about 10%) of non-flagellin polypeptide, and most preferably less than about 5%> non-flagellin polypeptide. When the flagellin polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

The language "substantially free of chemical precursors or other chemicals" includes preparations of flagellin polypeptide in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of flagellin polypeptide having less than about 30% (by dry weight) of chemical precursors or non-flagellin chemicals, more preferably less than about 20% chemical precursors or non-flagellin chemicals, still more preferably less than about 10% chemical precursors or non-flagellin chemicals, and most preferably less than about 5% chemical precursors or non-flagellin chemicals.

The invention also includes fragments of a flagellin polypeptide that includes various amounts of the amino terminus of a flagellin polypeptide. For example, the invention includes a polypeptide that includes at least seven amino acid residues present in amino acids 1 to 156 of SEQ ID NO:l. More preferably, the polypeptide includes, e.g., at least 15, 20, 25, 30, 50, 75, 100, 125, 140 or more amino acids of SEQ ID NO.l. Preferably, the polypeptide that includes the flagellin polypeptide fragment is less than 160 amino acids in length. For example, the polypeptide can include less than 157, 150, 125, 115, 100, 50, or 25 or fewer amino acids.

Also within the invention are polypeptides whose sequence differs from those of a wild-type flagellin polypeptide. For example, the amino acid sequence of the polypeptide can be at least 85% identical to at least 50 contiguous amino residues present in amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide. More preferably, the polypeptide is at least 90%), 95%, 98%> or even 99% to at least 50 contiguous amino acid residues of a flagellin polypeptide (e.g., SEQ ID NO:l). In other embodiments, the polypeptide is at least 85% (e.g., 95%, 98%) or even 99% or more identical to at least 75 contiguous amino acids of a flagellin polypeptide (e.g., SEQ ID NO: 1). In still further embodiments, the polypeptide is at least 85% (e.g., 95%o, 98% or even 99% or more identical to at least 100 contiguous amino acids of a flagellin polypeptide (e.g., SEQ ID NOT); or is at least 85% (e.g., 95%, 98% or even 99%o or more identical to at least 125 contiguous amino acids of a flagellin polypeptide (e.g., SEQ ID NO: 1).

If desired, a polypeptide derived from the amino terminus of a flagellin polypeptide can be provided as a chimeric protein or a fusion protein. As used herein, a flagellin "chimeric protein" or "fusion protein" comprises a flagellin polypeptide operatively linked to a non-flagellin polypeptide. An "flagellin polypeptide" refers to a polypeptide having an amino acid sequence corresponding to flagellin, whereas a "non-flagellin polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the flagellin protein, e.g., a protein that is different from the flagellin protein and that is derived from the same or a different organism. Within a flagellin fusion protein the flagellin polypeptide can correspond to all or a portion of a flagellin protein. In one embodiment, a flagellin fusion protein comprises at least one biologically active portion of a flagellin protein. In another embodiment, a flagellin fusion protein comprises at least two biologically active portions of a flagellin protein. In yet another embodiment, a flagellin fusion protein comprises at least three biologically active portions of a flagellin polypeptide. Within the fusion protein, the term "operatively linked" is intended to indicate that the flagellin polypeptide and the non-flagellin polypeptide are fused in-frame to each other. The non-flagellin polypeptide can be fused to the N-terminus or C-terminus of the flagellin polypeptide.

In yet another embodiment, the fusion protein is a GST-flagellin fusion protein in which the flagellin sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant flagellin.

In another embodiment, the fusion protein is a flagellin protein containing a heterologous signal sequence at its N-terminus. For example, the native flagellin signal sequence can be removed and replaced with a signal sequence from another protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion of flagellin can be increased through use of a heterologous signal sequence.

A flagellin polypeptide or fusion protein of the invention can be synthesized chemically using art-recognized techniques. Alternatively, a flagellin polypeptide can be produced by standard recombinant DNA techniques using nucleic acids that encode flagellin polypeptides or flagellin polypeptide fragments. An example of a nucleic acid encoding an amino terminal region of a flagellin polypeptide sequence is provided in Table 2.

ATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAAC AAATCCCAGTCCGCTCTGGGCACCGCTATCGAGCGTCTGTCTTCCGGTCTGCGTATC AACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAAC ATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAG ACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTG GCGGTTCAGTCTGCTAACGGTACTAACTCCCAGTCTGACCTTGACTCTATCCAGGCT GAAATCACCCAGCGTCTGAACGAAATCGACCGTGTATCCGGTCAGACTCAGTTCAAC GGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACGAC GGTGAAACTATT (SEQ ID NO : 2 )

The nucleic acid and polypeptide sequences of multiple bacterial flagellin sequences are known in the art. Accordingly, this information can be used to obtain nucleic acids encoding flagellin polypeptides using any method known in the art (e.g., by PCR amplification using synthetic primers hybridizable to the 3'- and 5 '-termini of the sequence and/or by cloning from a cDNA or genomic library using an oligonucleotide sequence specific for the given gene sequence).

An isolated nucleic acid molecule encoding a variant polypeptide homologous to the protein of SEQ ID NO:l can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:2, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

For recombinant expression of one or more flagellin polypeptides, the nucleic acid containing all or a portion of the nucleotide sequence encoding the peptide may be inserted into an appropriate expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted peptide coding sequence). In some embodiments, the regulatory elements are heterologous (i.e., not the native gene promoter). Alternately, the necessary transcriptional and translational signals may also be supplied by the native promoter for the genes and/or their flanking regions.

A variety of host-vector systems may be utilized to express the polypeptide coding sequence(s). These include, but are not limited to: (i) mammalian cell systems that are infected with vaccinia virus, adenovirus, and the like; (ii) insect cell systems infected with baculovirus and the like; (iii) yeast containing yeast vectors or (iv) bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

Promoter/enhancer sequences within expression vectors may utilize plant, animal, insect, or fungus regulatory sequences, as provided in the invention. For example, promoter/enhancer elements can b used from yeast and other fungi (e.g., the GAL4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter). Alternatively, or in addition, they may include animal transcriptional control regions, e.g., (i) the insulin gene control region active within pancreatic β-cells (see, e.g., Hanahan, et a , 1985. Nature 315: 115-122); (ii) the immunoglobulin gene control region active within lymphoid cells (see, e.g., Grosschedl, et al, 1984. Cell 38: 647-658); (iii) the albumin gene control region active within liver (see, e.g., Pinckert, et al, 1987. Genes and Dev 1 : 268-276; (iv) the myelin basic protein gene control region active within brain oligodendrocyte cells (see, e.g., Readhead, et al, 1987. Cell 48: 703-712); and (v) the gonadotropin-releasing hormone gene control region active within the hypothalamus (see, e.g., Mason, et al, 1986. Science 234: 1372-1378), and the like.

Expression vectors or their derivatives include, e.g. human or animal viruses (e.g., vaccinia virus or adenovirus); insect viruses (e.g., baculovirus); yeast vectors; bacteriophage vectors (e.g., lambda phage); plasmid vectors and cosmid vectors. A host cell strain may be selected that modulates the expression of inserted sequences of interest, or modifies or processes expressed peptides encoded by the sequences in the specific manner desired. In addition, expression from certain promoters may be enhanced in the presence of certain inducers in a selected host strain; thus facilitating control of the expression of a genetically-engineered peptides. Moreover, different host cells possess characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation, and the like) of expressed peptides. Appropriate cell lines or host systems may thus be chosen to ensure the desired modification and processing of the foreign peptide is achieved. For example, peptide expression within a bacterial system can be used to produce an unglycosylated core peptide; whereas expression within mammalian cells ensures "native" glycosylation of a heterologous peptide.

Fusion proteins including flagellin polypeptide fragments can similarly be constructed using recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A flagellin-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the flagellin protein.

Antibodies to Polypeptides Containing Amino Terminal Flagellin Sequences

Also included in the invention are antibodies to fragments of flagellin polypeptides (including amino terminal fragments), as well as antibodies to fusion proteins containing flagellin polypeptides or fragments of flagellin polypeptides. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, e.g., polyclonal, monoclonal, chimeric, single chain, F_ab, F_ab, and F_(ab.₎₂ fragments, and an F_ab expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG,, IgG₂, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

An amino terminal flagellin polypeptide of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. Antigenic peptide fragments of the antigen for use as immunogens includes, e.g., at least 7 amino acid residues of the amino acid sequence of the amino terminal region, such as an amino acid sequence shown in SEQ ID NO:l, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of flagellin polypeptide that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of a flagellin polypeptide will indicate which regions of a flagellin protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J Mol Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.

Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.

Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immuno stimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffmity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

Monoclonal Antibodies

The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.

After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison. Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

Humanized Antibodies

The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non- human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323- 327 (1988); Verhoeyen et al., Science. 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al, 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

Human Antibodies

Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779- 783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)). Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immuno globulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.

F_ab Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of F_ab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F_ac fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F_(ab.₎₂ fragment produced by pepsin digestion of an antibody molecule; (ii) an F_ab fragment generated by reducing the disulfide bridges of an F_(ab,₎₂ fragment; (iii) an F_ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F fragments.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMBO J, 10:3655-3659.

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab'), bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al. J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64), Fc RII (CD32) and Fc RIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOT A, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl -4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

Effector Function Engineering

It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

Immunoconj ugates

The invention also pertains to immunoconj ugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconj ugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14-labeled l-isofhiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.

Pharmaceutical Compositions

The invention also includes pharmaceutical compositions containing amino terminal sequences of a flagellin polypeptide, as well as pharmaceutical compositions containing antibodies to the amino terminal region of a flagellin polypeptide. The compositions are preferably suitable for internal use and include an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers. The compounds are especially useful in that they have very low, if any toxicity.

In practice, the compounds or their pharmaceutically acceptable salts, are administered in amounts which will be sufficient to inhibit inflammatory conditions or disease and/or prevent the development of inflammation or inflammatory disease in animals or mammals, and are used in the pharmaceutical form most suitable for such purposes.

Preferred pharmaceutical compositions are tablets and gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

Administration of the active compounds and salts described herein can be via any of the accepted modes of administration for therapeutic agents. These methods include systemic or local administration, such as intravenous, intraperitoneal, intramuscular, intraventricular, subcutaneous, topical, sublingual, oral, nasal, parenteral, transdermal, and subcutaneous or topical administration modes.

Depending on the intended mode of administration, the compositions may be in solid, semi-solid or liquid dosage form, such as, for example, injectables, tablets, suppositories, pills, time-release capsules, powders, liquids, suspensions, or the like, preferably in unit dosages. The compositions will include an effective amount of active compound or the pharmaceutically acceptable salt thereof, and in addition, and may also include any conventional pharmaceutical excipients and other medicinal or pharmaceutical drugs or agents, carriers, adjuvants, diluents, etc., as are customarily used in the pharmaceutical sciences.

For solid compositions, excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound defined above may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.

Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc. The active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.

If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.

Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or solid forms suitable for dissolving in liquid prior to injection.

One approach for parenteral administration employs the implantation of slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.

The compounds of the present invention can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as an antiandrogenic agent.

The dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicated effects, will range between about 0.05 to 1000 mg/day orally. The compositions are preferably provided in the form of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient. Effective plasma levels of the compounds of the present invention range from 0.002 mg to 50 mg per kg of body weight per day.

Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.1% to 15%, w/w or w/v.

The compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta- lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

Any of the above pharmaceutical compositions may contain 0.1-99%, preferably 1- 70% of the flagellin polypeptide.

If desired, the pharmaceutical compositions can be provided with an adjuvant. Adjuvants are discussed above. In some embodiments, adjuvants can be used to increase the immunological response, depending on the host species, include Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Generally, animals are injected with antigen using several injections in a series, preferably including at least three booster injections.

Methods of inhibiting gram negative-mediated infections

The flagellin polypeptides (as well as nucleic acids encoding these polypeptides), antibodies, and pharmaceutical compositions described herein can be used to inhibit in a subject an infection associated with a gram negative bacteria. The compositions disclosed herein can additionally be used to prevent a gram negative infection in a subject. Preferably, the subject is at risk for developing an infection associated with a gram negative bacteria. Alternatively, or in addition, the compositions can be used to treat a condition or disease associated with a gram negative bacterial infectionin a subject. The subject can be, e.g., a mammal, such as a human, a non-human primate, horse, cow, dog, cat, mouse, rat, pig, goat, sheep, guinea pig, hamster or rabbit.

Examples of gram negative bacteria whose infections can be inhibited or treated include, e.g., motile gram negative pathogens such as Escherichia coli, Yersinia spp., Salmonella spp., Shigella spp., Campylobacter spp., Yersinio spp., Pseudomonas spp., Proteus spp., Citrobacter spp. Conditions or diseases associated with gram negative bacterial infections include, e.g., peritonitis, pneumonia, bacteremia, sepsis, cystitis, urethritis, pyelonephritis, pericarditis, meningitis, endocarditis, osteomyelitis, cellulitis, endopthalmitis, sinusitis, periorbital cellulitis, pyogenic arthritis, encephalitis, hepatic abscess, pancreatitis, wound infection, burns, major surgery, gingivitis, soft- tissue abscess, otitis media, otitis externa, tonsillitis, adenoiditis, cholecystitis, typhlitis, perirectal abcess, fistulae, and others.

Assays and conditions for determining the efficacy of treatment of gram negative bacterial infections, as well as conditions associated with these infections are known in the art. These typically include bacterial culture and vital staining of tissues and bodily fluids, measurement of circulating white blood cell populations, and detection of fever.

The invention will be further illustrated in the following non-limiting examples.

Example 1: Antiserum produced in response to immunization with the N-terminal domain of recombinant Salmonella muenchen flagellin has broad spectrum immunoreactivity with flagellins from enteric gram negative bacilli

Serum was collected from New Zealand rabbits immunized with a GST fusion of the N-terminus of recombinant S. muenchen flagellin, corresponding to amino acids 1-156. The antigen was prepared by expression of a cDNA clone obtained by PCR amplification of DNA from Salmonella muenchen utilizing a sense primer designated IS (5' CGCGGATCCCAATG-GCACAAGTCATTAATACAAACA), and an anti-sense primer designated 468A (5' TCCGCTCGAGTTAAATAGTTTCACCGTCGTTGGCACC). Underlined nucleotides represent adaptor sequences added to the ends of primers to maintain proper reading frame and facilitate cloning - Bam HI recognition sites on sense primers and Xho I sites on antisense primers. Template DNA for PCR reactions was plasmid CL402, a clone of pBR322 containing a 3.8 kb EcoR I fragment of S. muenchen chromosomal DNA that harbors the 1.5 kb flagellin gene. PCR-generated flagellin DNA were digested with Bam HI plus Xho I, gel purified, and subcloned into the Bam Hl/Xho I sites at the 3' end of the GST gene in expression vector pGEX-5X-2 (Pharmacia Biotech, Piscataway, NJ). The correct reading frame and integrity of subcloned DNA was verified by DNA sequence analysis.

Antiserum was purified over an affinity column produced by coupling recombinant His-tagged S. muenchen full-length flagellin. Eluted antibodies were concentrated and used to probe Western blots of electrophoresed whole cell lysates and recombinant His-tagged flagellins cloned and expressed by the PI.

The results are shown in FIGS. 1 A-IC. Anti-flagellin Abs recognized a broad spectrum of motile enteric gram negative bacilli. The results are shown in FIGS. lA-lC. FIG. 1 A shows Western blot analysis of recombinant flagellins. Purified flagellin proteins (45 ng) were separated on a 10% SDS-polyacrylamide gel and proteins were transferred to a nitrocellulose filter by electroblotting. The blot was probed with affinity purified rabbit polyclonal antibodies raised against N-terminus Salmonella muenchen flagellin protein. Lane. 1 : Escherichia coli; lane. 2: Salmonella muenchen; lane. 3: Serratia marcescens; lane. 4: Pseudomonas aeruginosa; lane. 5: Proteus mirabilis; lane.6: Proteus vulgaris. The fast migrating protein in lane 2 is a truncated product.

FIG. IB shows the results of immuno blot analysis of bacterial proteins with affinity purified flagellin antibodies. Bacterial strains were grown over night in Nutrient Broth medium at 37°C. Whole cell extracts equivalent to l xlO⁷ cells were fractionated on a 10%> SDS-polyacrylamide gel and probed with flagellin antibodies (as mentioned in Fig. 1 A). The samples were: Recombinant flagellin (Salmonella muenchen, lane 1), Escherichia coli non- pathogenic (lane 2), Escherichia coli enteropathogenic (lane3), Salmonella typhimurium (lane 4), Serratia marcescens (lane 5), Pseudomonas aeruginosa (lane 6), Proteus mirabilis (lane 7), Proteus vulgaris (lane 8). The slow migrating protein (in lanes 2-8) is a cross-reacting bacterial protein. (C) Immuno blot analysis of bacterial proteins with affinity purified flagellin antibodies. Experimental conditions were same as in Fig. 1 A except bacterial strains were grown in LB medium. The samples were: Salmonella typhimurium (lane 1), Serratia marcescens (lane 2), Klebsiella pneumoniae (lane 3), Pseudomonas aeruginosa (lane 4), Proteus mirabilis (lane 5), Proteus vulgaris (lane 6). The slow migrating protein (in lanes 1-6) is a cross-reacting bacterial protein.

The absence of immunoreactivity to Pseudomonas aeruginosa was not entirely unexpected, given that this non-enteric GNB diverges slightly more in sequence in the N- terminal domain of flagellin than enteric GNB's. Nonetheless, there are several regions of near total alignment between Pseudomonas sp. and Salmonella muenchen; thus, monoclonal antibodies generated in response to immunization with the N-terminus of 5. muenchen flagellin may bind to epitopes immunoreactive with P. aeruginosa flagellin. Indeed, the polyclonal antibodies raised against RINSA peptide (amino acids 40-58) or monoclonal antibodies raised against N-teminus Salmonella muenchen flagellin immunoreacted with Pseudomonas flagellin protein from whole cell extract. One possible explanation for this discrepancy is that the cloned gene may have some mutations, as we did not sequence entire gene.

Example 2. Pre-incubation of is. coli with purified polyclonal antiserum immunoreactive with the N-terminal domain of recombinant Salmonella muenchen flagellin reduces infectivity in a murine model of urinary tract infection

Female, 5-6 week old, Balb/c female mice were randomized into 2 study groups (n=5 mice per group). The challenge inoculums of Escherichia coli (strain 616UB) were grown from frozen stocks by two successive passages in tryptose broth, one for 48 hours, and the second for 24 hours immediately prior to mouse inoculation. On the day of infection, bacteria were harvested from broth by centrifugation, washed once with PBS, and resuspended in PBS at 2 x 10¹⁰ per ml. The bacteria were then diluted 1 : 1 with purified anti-N terminal flagellin Ab (described in C.6) or saline, and incubated for 15 minutes at room temperature. The suspensions were then drawn into a 100 L syringe attached to a 30 G needle fitted with a 0.5 cm piece of Intramedic PE-10 polyethylene tubing. Mice anesthetized with ether were inoculated intravesically per urethra with 10 L of the suspension, resulting in an inoculum of 10⁸ bacteria per animal. 24 hours after intravesical inoculation, animals were euthanized and necropsied for evidence of infection. Mice were evaluated for urogenital infection by standard culture techniques: mouse bladder was aseptically removed, weighed, and homogenized with glass homogenizers. Homogenates were centrifuged at low speed to remove large tissue debris, and dilutions of the resultant supernates were plated onto EMB Levine agar (Fisher Scientific, Itasca, IL). Colony-forming units were calculated for individual animals.

The results are shown in FIG. 2. Exposure ofE. coli to purified antiserum directed against the N-terminus of flagellin resulted in a 78%> reduction in bladder infectivity (p<0.03). Antiserum raised in rabbits immunized with a GST fusion of the N-terminus of S. muenchen flagellin reduces infectivity in a murine model of UTI. E. coli were incubated with purified anti- N-terminal flagellin antisera and then instilled into Balb/c female mice via a transurethral route into the bladder. Bladders were removed after 24 hours and cultured to determine infectivity. Exposure of E. coli to purified antiserum directed against the N-terminus of flagellin resulted in a 78% reduction in bladder infectivity. These data demonstrate that immunization against the N-terminus of flagellin provides protection against gram negative infection. Moreover, antisera raised against Salmonella muenchen was found to be protective in an in vivo model of E. coli infection; thereby demonstrating the broad-spectrum of activity offered by this therapeutic approach.

Example 3. Passive immunization with lapine polyclonal anti-S. muenchen N-terminal flagellin antiserum protects against infectivity, inflammation, and mortality in an acute murine model of Serratia marcescens pneumonia.

Male, 8 week old, Balb/c mice were randomized into 2 study groups («=8 mice per group). Group 1 mice received a 20 mL/kg intravenous bolus of pre-immune lapine sera. Group 2 mice received an intravenous bolus of an identical volume of W2. W2 was obtained from New Zealand rabbits immunized with a GST fusion protein containing the N-terminus of Salmonella muenchen. Rabbits received a primary immunization, in complete Freund's adjuvant, followed by two booster immunizations in incomplete Freund's adjuvant.

The challenge inoculum of Serratia marcescens was harvested from broth by centrifugation, washed three times with PBS, and resuspended in PBS at 10⁷ per ml. 1 h after administration of the antiserum (or PBS vehicle control), 100 L of the bacterial suspension was drawn into a 1 mL syringe attached to a 27 G needle and injected under sterile conditions directly into the trachea of anesthetized mice (following a sterile dissection of the neck to expose the proximal trachea). The soft tissue defect overlying the trachea was immediately brought together with glue and the animals were allowed to recover. Mice had free access to food and water. After 24h, mice were sacrificed, lungs excised for analysis, and blood obtained by cardiac puncture for culture (3). In the group receiving pre-immune serum, survival was 0%> at 24 hours.

In an LD₂₀₀ model of Serratia marcescens induced acute pneumonia, passive immunization with the antiserum eliminated mortality, reduced bacterial cfu in the lung by 90%) (p<0.001), decreased pulmonary neutrophil infiltration by 95% (p<0.001), and reduced the incidence of bacteremia by 50% (p<0.05). Example 4. Active immunization with S. muenchen N-terminal flagellin protects against mortality in an acute murine model of Serratia marcescens peritonitis.

Male, 8 week old, Balb/c mice received a primary immunization with a His-tagged recombinantly expressed S. muenchen N-terminal flagellin in complete Freund's adjuvant, followed by two booster immunizations in incomplete Freund's adjuvant. Recombinant plasmid containing His-tag sequences at its N-terminus was constructed by inserting Bam Hl/Xho I PCR fragment (as described in example 1) coding for N-terminus flagellin protein at Bam Hl/Xho I sites of expression vector pET 30 (invitrogen).At 16 weeks of age, immunized mice, and naϊve age-matched controls, were exposed to an i.p. challenge of a LD₁₀₀ dose of Serratia marcescens (4χl0⁸ cfu in 0.5 mL of PBS) («=10 mice per group). All naϊve mice died within 24 hours. All immunized mice survived and showed no signs of illness at 24 hours post-challenge.

Example 5. Active immunization with S. muenchen N-terminal flagellin protects against mortality in an acute murine model of Serratia marcescens pneumonia.

Male, 8 week old, Balb/c mice received a primary immunization with a His-tagged recombinantly expressed S. muenchen N-terminal flagellin in complete Freund's adjuvant, followed by two booster immunizations in incomplete Freund's adjuvant. At 16 weeks of age, immunized mice, and naϊve age-matched controls, were exposed to an acute intratracheal challenge of Serratia marcescens (10⁷ cfu), administered exactly as described in Section B.4.9. (n=10 mice per group). Nine of ten naϊve mice died within 24 hours. In contrast, nine of 10 immunized mice survived and showed no signs of illness at 24 hours post-challenge.

Other Embodiments

Other embodiments are within the claims.

Claims

What is claimed is:

1. A purified flagellin polypeptide fragment less than 160 amino acids in length, wherein said polypeptide has an epitope that binds to an antibody that neutralizes a gram- negative bacterial infection.

2. The polypeptide of claim 1, wherein said polypeptide is at least 85% homologous to the corresponding amino acid sequence of amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide.

3. The polypeptide of claim 1, wherein said polypeptide is at least 90%> homologous to the corresponding amino acid sequence of amino acids 1 to 156 of & Salmonella muenchen flagellin polypeptide.

4. The polypeptide of claim 1, wherein said polypeptide is at least 95% homologous to the corresponding amino acid sequence of amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide.

5. The polypeptide of claim 1, wherein the amino acid sequence of said polypeptide comprises at least seven amino acid residues present in amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide.

6. The polypeptide of claim 1, wherein the amino acid sequence of said polypeptide comprises at least 15 contiguous amino acid residues present in amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide.

7. The polypeptide of claim 1, wherein the amino acid sequence of said polypeptide comprises an amino acid sequence at least 95% identical to at least 50 contiguous amino residues present in amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide.

8. The polypeptide of claim 1, wherein the amino acid sequence of said polypeptide comprises an amino acid sequence at least 95% identical to at least 100 contiguous amino residues present in amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide.

9. The polypeptide of claim 1, wherein said Salmonella muenchen flagellin polypeptide comprises SEQ ID NO: 1.

10. The polypeptide of claim 1, wherein the amino acid sequence of said polypeptide comprises the amino acid sequence of a fragment of a flagellin polypeptide selected from the group consisting of a Salmonella spp., an Aeromonas spp., a Yersinia spp., a Proteus spp., a Serratia spp. a Pseudomonas spp. and a Vibrio spp.

11. A fusion polypeptide comprising the polypeptide of claim 1.

12. The fusion polypeptide of claim 1 1 , wherein said fusion protein comprises a glutathione-S-transferase (GST) polypeptide or a plurality of contiguous histidine residues.

13. A nucleic acid encoding the polypeptide of claim 1.

14. A vector comprising the nucleic acid of claim 13.

15. A cell containing the vector of claim 14.

16. An antibody that binds specifically to a flagellin polypeptide fragment less than 160 amino acids in length.

17. The antibody of claim 16, wherein said antibody binds to a flagellin polypeptide fragment that is at least 85% homologous to the corresponding amino acid sequence of amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide.

18. The antibody of claim 16, wherein said antibody binds to a flagellin polypeptide fragment comprising at least seven amino acid residues present in amino acids 1 to 156 of a Salmonella muenchen flagellin polypeptide.

19. The antibody of claim 16, wherein said antibody binds to a flagellin polypeptide fragment comprising the amino acid sequence of SEQ ID NO:l.

20. The antibody of claim 16, wherein said antibody is a neutralizing antibody.

21. The antibody of claim 16, wherein said antibody is a polyclonal antibody.

22. The antibody of claim 16, wherein said antibody is a monoclonal antibody.

23. The monoclonal antibody of claim 22, wherein said monoclonal antibody is selected from the group consisting of a mouse monoclonal antibody, a murine monoclonal antibody, and a humanized monoclonal antibody.

24. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, further comprising an adjuvant.

26. A pharmaceutical composition comprising the antibody of claim 16 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, further comprising an adjuvant.

28. A method of preventing an infection associated with a gram negative bacterium in a subject, the method comprising administering to said subject the polypeptide of claim 1 to said subject.

29. The method of claim 28, wherein said subject is a mammal.

30. The method of claim 29, wherein said mammal is selected from the group consisting of a human, horse, cow, dog, cat, mouse, rat, pig, goat, sheep, and rabbit.

31. A method of preventing an infection associated with a gram negative bacterium in a subject, the method comprising administering to said subject the antibody of claim 16 to said subject.

32. The method of claim 31 , wherein said subject is a mammal.

33. The method of claim 31 , wherein said mammal is selected from the group consisting of a human, horse, cow, dog, cat, mouse, rat, pig, goat, sheep, and rabbit.

34. A method of preventing an infection associated with a gram negative bacterium in a subject, the method comprising administering to said subject the polypeptide of claim 1 to said subject.

35. The method of claim 34, wherein said subject is a mammal.

36. The method of claim 35, wherein said mammal is selected from the group consisting of a human, horse, cow, dog, cat, mouse, rat, pig, goat, sheep, and rabbit.

37. A method of preventing an infection associated with a gram negative bacterium in a subject, the method comprising administering to said subject the antibody of claim 16 to said subject.

38. The method of claim 37, wherein said subject is a mammal.

39. The method of claim 37, wherein said mammal is selected from the group consisting of a human, horse, cow, dog, cat, mouse, rat, pig, goat, sheep, and rabbit.