Escherichia coli nhaa membrane protein drug target and immunogenic compositions therefrom

Abstract

The present disclosure is directed to a novel extraintestinal pathogenic Escherichia coli (ExPEC) drug target, NhaA. Mutant ExPEC bacteria comprising a disruption of the endogenous nhaA gene are provided, as are immunogenic compositions therefrom and methods of inducing an immune response against ExPEC in a subject.

Description

Agent Ref. P14443WO00 1 TITLE: ESCHERICHIA COLI NHAA MEMBRANE PROTEIN DRUG TARGET AND IMMUNOGENIC COMPOSITIONS THEREFROM REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is herein incorporated by reference in its entirety. Said XML copy, created on June 29, 2023, is named “P14443WO00_SequenceListing.xml” and is 55,022 bytes in size. TECHNICAL FIELD

[0002] The present disclosure relates generally to extraintestinal pathogenic Escherichia coli drug targets and related immunogenic compositions. BACKGROUND

[0003] Extraintestinal pathogenic Escherichia coli (ExPEC) causes lethal bloodstream infections in humans and animals. (ExPEC) breaches a sterile barrier to cause severe extraintestinal diseases in all age groups of humans and animals. In humans, ExPEC is responsible for most urinary tract infections and a large proportion of bloodstream infections. The ExPEC responsible for poultry infections is avian pathogenic E. coli (APEC), which causes diverse local and systemic infections in poultry, including chickens, turkeys, ducks, and many other avian species. APEC causes significant economic losses in the poultry industry. A common feature of ExPEC is its ability to cause bloodstream infection leading to septicemia, and some strains, including those of human and avian origin, exhibit zoonotic potential.

[0004] To survive and proliferate in the blood, ExPEC must be able to resist the multiple innate defense systems present in the blood including the effects of phagocytes, the complement system, lysozyme, antimicrobial peptides and coagulation factors VII, IX and X. As part of the innate immune system, the complement system plays a pivotal role in the recognition and direct clearance of invading microbes and aberrant host cells. Furthermore, classical (CP), lectin (LP), and alternative pathways (AP) are triggered via the recognition of antigen-bound IgG (by the C1 complex), conserved sugar residues on bacterial surfaces, and spontaneous hydrolysis of C3, respectively, to activate the complement system. Following the formation of the C3 convertase, the three complement pathways converge, and C3 convertase cleaves the central complement component C3 into C3a and C3b. Moreover, C3b participates in C5 convertase formation, which cleaves C5 and initiates sequential deposition of the remaining complement components to form the C5b-9 membrane attack complex (MAC). These convertase-generated MAC pores in the outer membrane (OM) trigger inner membrane (IM) damage and directly kill microorganisms. Complement-mediated lysis of susceptible bacteria isAgent Ref. P14443WO00 2 reportedly enhanced by antimicrobial peptides, proteases and lysozyme. However, MAC damages bacterial IM and kills bacteria without depending on lysozyme.

[0005] Multiple factors contributing to resistance to complement-mediated killing have been identified in pathogenic E. coli. Furthermore, mechanisms promoting resistance include the production of protective polysaccharide layers, interference with the complement cascade, and the expression of factors that contribute to the structural integrity of the cell envelope. Notably, K- and O-antigen capsules, lipopolysaccharides, extracellular polysaccharides, and colanic acid contribute to ExPEC strain survival in the serum. Moreover, membrane proteins (OmpA, NlpI and Prc) contribute to E. coli evasion of complement-mediated serum killing. Additionally, two plasmid-encoded OM lipoproteins, TraT and Iss, have been suggested to interfere with the MAC activity. However, the use of these virulence factors in the treatment and prevention of ExPEC infection has not been shown to be feasible.

[0006] Accordingly, there remains a need in the art for the identification of ExPEC pathogenic mechanisms and the development of effective vaccines for the control of ExPEC-related diseases. SUMMARY

[0007] The present disclosure encompasses immunogenic compositions comprising an NhaA protein of an extraintestinal pathogenic Escherichia coli (ExPEC) bacteria and a pharmaceutically acceptable carrier. The present disclosure also encompasses immunogenic compositions comprising mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria and a pharmaceutically acceptable carrier. In some embodiments, the mutant ExPEC bacteria comprises a disruption of the endogenous nhaA gene. In certain embodiments, the ExPEC bacteria comprises avian pathogenic E. coli (APEC) bacteria. In some embodiments, the mutant ExPEC bacteria is a live, attenuated bacteria.

[0008] In certain embodiments the disruption of the nhaA gene or NhaA protein may comprise deletion of the nhaA gene from the bacterial genome. The disruption of the endogenous nhaA gene may result in complete inactivation of the NhaA protein. This inactivation can attenuate the virulence of the mutant ExPEC bacteria as compared to wild-type ExPEC bacteria.

[0009] In some embodiments, the nhaA gene is not deleted but is mutated such that virulence is attenuated as compared to wild-type ExPEC bacteria. The disruption of the endogenous nhaA gene may comprise at least one mutation to loop-2, loop-3, loop-4, loop-5, loop-6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein. The at least one mutation may comprise a site-mutation or may comprise a knock-out mutation of the loop. The mutation can comprise at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO: 2. In some embodiments, the at least one mutation comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO: 2.Agent Ref. P14443WO00 3

[0010] Methods of inducing an immune response against ExPEC in a subject are also provided. In some embodiments, the method comprises administering to the subject an NhaA protein of an extraintestinal pathogenic Escherichia coli (ExPEC) bacteria and a pharmaceutically acceptable carrier. In some embodiments, the method comprises administering to the subject an immunogenic composition comprising a mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene. In certain embodiments, administration of the mutant ExPEC bacteria immunizes the subject against a disease, condition, or pathology caused by ExPEC bacteria.

[0011] The present disclosure also provides mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene or NhaA protein. The disruption of the endogenous nhaA gene may comprise deletion of the nhaA gene or mutation of the nhaA gene. In certain embodiments, disruption of the endogenous nhaA gene comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO: 2. The at least one mutation may comprise an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO: 2.

[0012] These and / or other objects, features, advantages, aspects, and / or embodiments will become apparent to those skilled in the art after reviewing the following brief and detailed descriptions of the drawings. The present disclosure encompasses (a) combinations of disclosed aspects and / or embodiments and / or (b) reasonable modifications not shown or described. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the disclosure. In some instances, embodiments of the disclosure can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the disclosure. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the disclosure.

[0014] FIGS.1A-1E show that nhaA deletion significantly attenuates ExPEC XM's virulence in chick and mouse models. (1A) WT, ∆nhaA mutant, and ∆nhaA-complemented strains (5 × 107CFU) were inoculated into eight 7-day-old chicks per group, and the mortality was monitored for 1 week. The virulence of the ∆nhaA mutant was significantly attenuated compared with that of the WT strain (P < 0.001 by log-rank (Mantel-Cox) test). (1B) nhaA deletion caused a significant decrease of the bacterial load in the blood of infected chicks at 12 h post-infection (P < 0.001 by Student’s t-test). The dotted line represents the blood's lower limit of detection, 50 CFU / mL blood. (1C) WT, ∆nhaA mutant, andAgent Ref. P14443WO00 4 ∆nhaA-complemented strains (5 × 107CFU) were inoculated into eight 6-week-old mice per group, and the mortality was monitored. The virulence of the ∆nhaA mutant was significantly attenuated compared with that of the WT strain (P < 0.001 by log-rank (Mantel-Cox) test). (1D) nhaA deletion significantly decreased the bacterial load in the blood of infected mice at 12 h post-infection (P < 0.01 by Student’s t-test). The dotted line represents the blood's lower limit of detection, 50 CFU / mL blood. (1E) Growth curves for the WT, ΔnhaA and ΔnhaA-complemented strains administered in M9 minimal medium supplemented with glucose as energy substrates were examined by monitoring the OD600of each sample every 1 h.

[0015] FIGS. 2A-2E show identification of protein loops and amino acid residues of NhaA contributing to ExPEC virulence. The amino acid residues of NhaA are also presented in SEQ ID NO: 2 (2A) The two-dimensional model of E. coli NhaA. Triangles represent mutation sites that inactivate the antiporter; squares represent sites where mutations shift the pH profile; circles represent sites where mutations affect the Km for Na+and Li+. (2B and 2C) Survival curve of chicks infected with loop- and site-mutation strains (n = 8). (2D and 2E) Survival rates of mice infected with loop- and site-mutation strains at 24 h post-infection (n = 8). Significant differences were evaluated by log-rank (Mantel-Cox) test, and the asterisks *** indicate P < 0.001, **, P < 0.01, *, P < 0.05.

[0016] FIGS.3A-3D show the effects of nhaA deletion on the envelope integrity of ExPEC XM. (3A) EDTA and SDS sensitivity assays of WT, ΔnhaA, and ∆nhaA-complemented strains. The bacterial cultures were adjusted to OD600 ≈ 0.4 with double distilled water, and ten-fold serial dilution was performed on LB, LB with 1 mM EDTA, 2 mM EDTA, or 2% SDS agar plates. (3B and 3C) Growth curves for the WT, ΔnhaA, and ΔnhaA-complemented strains inoculated in different culture media were examined by monitoring the OD600of each sample every 1 h. (3D) RNase leakage assays. The bacteria were grown on an RNase test agar plate. The pink halos around the colonies indicate the leakage of periplasmic RNase into the agar.

[0017] FIGS. 4A-4D show that nhaA gene deletion reduces the resistance of ExPEC to serum bactericidal effects. (4A) Survival capacity of WT, ΔnhaA, and ΔnhaA-complemented strains incubated in 90% avian serum at an initial inoculation dose of 5 × 106CFU / mL for 4 h. (4B) Survival capacity of WT, ΔnhaA and ΔnhaA-complemented strains incubated in 90% human serum at an initial inoculation dose of 5 × 106CFU / ml for 4 h. Dotted line represents the blood lower limit of detection, 50 CFU / mL blood. Data are presented as the mean ± standard deviation. Significant differences were evaluated using the Student's t-test (4A and 4B), and the asterisks *** indicate P < 0.001. ns, not significant. (4C) (Left panel) The morphology of WT, ΔnhaA, and ΔnhaA-complemented strains cultured in LB medium. (Right panel) The morphology of WT, ΔnhaA, and ΔnhaA-complemented strains cultured in 90% avian serum. Cells were prepared as described in Materials and Methods for visualization by laser confocal microscopy (scale bars: 5 μm). (4D) The morphology of WT, ΔnhaA,Agent Ref. P14443WO00 5 and ΔnhaA-complemented strains cultured in LB medium (left panel) and 90% avian serum for 2 h (right panel) and visualized by transmission electron microscopy (scale bars: 1 μm).

[0018] FIGS.5A-5B show that NhaA contributes to the resistance to complement-mediated bacterial killing. (5A) Survival capacity of WT, ΔnhaA, and ΔnhaA-complemented strains incubated in 90% NAS and 90% HIAS at an initial inoculation dose of 5 × 106CFU / mL for 4 h. (5B) Survival capacity of WT, ΔnhaA and ΔnhaA-complemented strains incubated in 90% NHS and 90% HIHS at an initial inoculation dose of 5 × 106CFU / mL for 4 h. Dotted line represents the blood lower limit of detection, 50 CFU / mL blood. Data are presented as the mean ± standard deviation. Significant differences were evaluated using the Student's t-test, and the asterisks *** indicate P < 0.001.

[0019] FIGS. 6A-6C show that NhaA impairs C3b and MAC depositions through classical and alternative pathways. C3b and C9 depositions on ΔnhaA and WT strains in 50% human serum for various periods. (6A) C3b deposition levels on ΔnhaA and WT after incubation with 50% normal human serum for the indicated periods (top panel). Flow cytometry histogram of C3b deposition on the bacteria after 2 h incubation in 50% NHS (bottom panel). (6B) C9 deposition levels on ΔnhaA and WT after incubation with 50% normal human serum for the indicated periods (top panel). Flow cytometry histogram of C9 deposition on the bacteria after 2 h incubation in 50% NHS (bottom panel). The data are presented with mean fluorescence intensity (MFI). The WT strains incubated with FITC- conjugated secondary antibodies were used as the control group. The results are expressed as the means ± standard deviations, and the data represent three independent experiments performed in triplicate. Significant differences were evaluated using the Student's t-test, and the asterisks ***indicate P < 0.001, and **, P < 0.01. (6C) The serum survival of ΔnhaA and WT strains after 3 h incubation in 50% NHS and NHS with the classical (CP--HS), alternative (AP--HS), or lectin pathway inhibited (LP- -HS) sera (top panel). Flow cytometry histogram of C3b deposition on the bacteria after 2 h incubation in different sera (bottom panel). Data are presented as the mean ± standard deviation. Significant differences were evaluated using the Student's t-test, and the asterisks ** indicate P < 0.01, * P < 0.05.

[0020] FIGS. 7A-7B show that NhaA plays an important role in ExPEC resistance to serum-killing activity and ExPEC virulence. (7A) The serum survival of WT and ΔnhaA, Δiss, Δprc, ΔompA, and ∆nlpI strains initial inoculation dose of 5 × 106CFU / mL in 90% NAS for 4 h incubation. The virulence of ΔnhaA and Δprc mutant was significantly attenuated compared with that of the WT strain (P < 0.01 by Student's t-test). Data are presented as the mean ± standard deviation. Significant differences were evaluated using the Student's t-test, and the asterisks ** indicate P < 0.01. (7B) WT, ΔnhaA, Δiss, Δprc, ΔompA, and ∆nlpI strains (5 × 107CFU) were inoculated into eight 7-day-old chicks per group, and the mortality was monitored for 1 week. Significant differences were evaluated by log-rank (Mantel-Cox) test, and the asterisks *** indicate P < 0.001. **, P < 0.01. ns, not significant.Agent Ref. P14443WO00 6

[0021] FIGS. 8A-8B show that nhaA is important for other ExPEC prototype strains' resistance to serum-killing activity and their virulence. (8A) The serum survival of WT and ΔnhaA in different ExPEC strains at an initial incubation dose of 5 × 106CFU / mL in 50% NHS for 4 h. Data are presented as the mean ± standard deviation. Significant differences were evaluated using the Student's t-test, and the asterisks *** indicate < 0.001, **, P < 0.01. (8B) ΔnhaA of CFT073, RS218, UTI89, and their WT strains were inoculated into eight 6-week-old mice per group, and the mortality was monitored for 1 week. Significant differences were evaluated using the log-rank (Mantel-Cox) test, and the asterisks ***indicate P < 0.001, **, P < 0.01, *, P < 0.05. DETAILED DESCRIPTION

[0022] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosure pertain. The definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed disclosure. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments without undue experimentation, but the preferred materials and methods are described herein. In describing and claiming the embodiments, the following terminology will be used in accordance with the definitions set out below.

[0023] It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms "a," "an" and "the" can include plural referents unless the content clearly indicates otherwise. Further, all units, prefixes, and symbols may be denoted in its SI accepted form. Numeric ranges recited within the specification are inclusive of the numbers within the defined range. Throughout this disclosure, various aspects are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0024] As used herein, the term “and / or”, e.g., “X and / or Y” shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning, e.g., A and / or B includes the options i) A, ii) B or iii) A and B.

[0025] It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely,Agent Ref. P14443WO00 7 various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0026] The term "about," as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents to the quantities.

[0027] “Antibodies” refers to polyclonal and monoclonal antibodies, chimeric, and single chain antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library. With respect to antibodies, the term, “immunologically specific” refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

[0028] An “attenuated” ExPEC bacteria as used herein refers to an ExPEC bacteria which is capable of infecting and / or replicating in a susceptible host but is non-pathogenic or less-pathogenic to the susceptible host. For example, the attenuated bacteria may cause no observable / detectable clinical manifestations, or less clinical manifestations, or less severe clinical manifestations, or exhibit a reduction in bacteria replication efficiency and / or infectivity, as compared with the related field isolated / wild-type strains. The clinical manifestations of ExPEC infection may include, without limitation, diarrhea, stomach cramping, abdominal pain or tenderness, nausea, vomiting, lower back pain, urinary urgency and / or frequency, chills, and headache. Further, ExPEC infection may manifest, for example, as a urinary tract infection (UTI), bacteremia, meningitis, and septicemia.

[0029] The term “disruption” as used herein refers to a mutation in a wild-type gene that results in a gene that is not wild-type, including deletions, knock-out mutations, and loss-of-function mutations.

[0030] An “immunogenic or immunological composition” refers to a composition of matter that comprises at least one antigen, which elicits an immunological response in the host of a cellular and / or antibody-mediated immune response to the composition or vaccine of interest. Usually, an “immune response” or “immunological response” includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and / or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction orAgent Ref. P14443WO00 8 lack of clinical signs normally displayed by an infected host, a quicker recovery time and / or a lowered duration or bacterial titer in the tissues or body fluids or excretions of the infected host compared to a healthy control. Preferably said reduction in symptoms is statistically significant when compared to a control. In some embodiments, the immunogenic compositions may be used as a “vaccine”. The term “vaccine”, as used herein, refers to an antigenic preparation used to produce immunity to a disease, in order to prevent or ameliorate the effects of infection. Vaccines are typically prepared using a combination of an immunologically effective amount of an immunogen together with an adjuvant effective for enhancing the immune response of the vaccinated subject against the immunogen.

[0031] The terms "include" and "including" when used in reference to a list of materials refer to but are not limited to the materials so listed.

[0032] As used herein, the terms “knockout”, “knockout mutation”, or “knockout mutant” refer to the targeted removal or inactivation of a specific gene within an organism’s genome. Knockouts can be accomplished through a variety of genetic engineering techniques known in the art, including, but not limited to homologous recombination, site specific nucleases, zinc-finger nucleases, transcription activator-like effect nucleases (TALENs), and CRISPR / Cas9. Knockout mutants may also be referred to herein as “null mutants”.

[0033] The term “mutant” as used in herein refers to organisms having at least one genomic alteration as compared to a wild-type organism. As used herein, “mutant” refers only to organisms comprising a mutation due to genetic engineering / modification and is not inclusive of organisms comprising a naturally occurring mutation.

[0034] As used herein, “a pharmaceutically acceptable carrier” or “pharmaceutical carrier” includes any and all excipients, solvents, growth media, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, inactivating agents, antimicrobial, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Such ingredients also include those that are safe and appropriate for use in veterinary applications. Pharmaceutically acceptable carriers are typically non-toxic, inert, solid or liquid carriers.

[0035] The term “subject” as used herein refers to any living being that would benefit from the compositions and methods described herein. For example, the subject may be an animal, including a human, avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, and porcine animal. Subjects may also be domesticated animals such as cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs, and the like. Avian animals includes poultry animals, such as chickens, turkeys, ducks, geese, guinea fowl, pigeons, ostrich, emu, partridge, pheasant, and the like. In certain embodiments, the subject is a human. In certain embodiments, the subject is a poultry animal.Agent Ref. P14443WO00 9

[0036] Immunogenic compositions will contain a “therapeutically effective amount” of the active ingredient, that is, an amount capable of eliciting an induction of an immunoprotective response in a subject to which the composition is administered. In the treatment and prevention of ExPEC infections, for example, a “therapeutically effective amount” would preferably be an amount that enhances resistance of the immunized subject to new infection and / or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by a subject infected with ExPEC, a quicker recovery time and / or a lowered bacterial count. Immunogenic compositions can be administered prior to infection, as a preventative measure against ExPEC. Alternatively, immunogenic compositions can be administered after the subject has already showed clinical manifestations of infection. Immunogenic compositions given after manifestations of ExPEC may be able to attenuate the infection, triggering a superior immune response than the natural infection itself.

[0037] The present disclosure provides for reduction of the incidence of and / or severity of clinical symptoms associated with ExPEC infection. Preferably, the severity and / or incidence of clinical symptoms in subjects receiving the immunogenic composition of the present disclosure are reduced at least 10% in comparison to subjects not receiving such an administration when both groups (subjects receiving and subjects not receiving the composition) are challenged with or exposed to infection by ExPEC. In some embodiments, the incidence or severity is reduced at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, wherein the subjects receiving the composition of the present disclosure exhibit no clinical symptoms, or alternatively exhibit clinical symptoms of reduced severity.

[0038] The term "weight percent," "wt. %," "percent by weight," "% by weight," and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, "percent," "%," and the like are intended to be synonymous with "weight percent," "wt. %," etc.

[0039] The methods and compositions may comprise, consist essentially of, or consist of the components and ingredients as well as other ingredients described herein. As used herein, "consisting essentially of" means that the methods and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed methods and compositions. Immunogenic Compositions

[0040] The present disclosure provides immunogenic compositions, suitable for use as vaccines and / or treatments against infection by extraintestinal pathogenic Escherichia coli (ExPEC) bacteria. TheAgent Ref. P14443WO00 10 immunogenic compositions according to the disclosure elicit a specific humoral immune response toward ExPEC comprising neutralizing antibodies.

[0041] The immunogenic and vaccine compositions of this disclosure are not, however, restricted to any particular type or method of preparation. These include, but are not limited to, infectious DNA vaccines (i.e., using plasmids, vectors or other conventional carriers to directly inject DNA into person), live vaccines, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, genetically engineered vaccines, etc.

[0042] In certain embodiments, the immunogenic composition comprises an NhaA protein of an ExPEC bacteria; and a pharmaceutically acceptable carrier. In certain embodiments, the immunogenic composition comprises a mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene; and a pharmaceutically acceptable carrier.

[0043] NhaA is an intramembrane protein encoded by the nhaA gene evolutionarily conserved in all kingdoms of life that regulates cellular ion homeostasis. The amino acid sequences of NhaA in all ExPEC strains, such as XM, UTI89, RS218, and CFT073, are 100% identical and 98% identical to that of E. coli K12. NhaA consists of 12 transmembrane helices connected by hydrophilic loops (see FIG. 2A). Moreover, it is organized into two functional regions: a pH sensor region and catalytic region containing an ion-binding site, and it is the main electrogenic sodium / hydrogen (Na+ / H+) antiporter. Using the site-directed mutagenesis approach, it was determined that residues such as Glu252, Glu241, and Val254 are essential in “pH sensor” functions; Asp65, Pro129, Thr132, and Asp133 are essential for the exchange of sodium / lithium (Na+ / Li+) and H+; Gly338, Ala127, and Glu252 are essential for pH sensing and exchange of Na+ / Li+and H+; and Asp163, Asp164, and Lys300 are essential for Na+ / H+activity and Li+binding. These amino acid residues are conserved in all ExPEC and commensal E. coli K12 strains.

[0044] Because the nhaA gene is conserved in all ExPEC strains, it is an ideal target for providing broad protection and / or treatment against all ExPEC variants, including, for example, uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), those isolates responsible for septicemia (SEPEC), avian pathogenic E. coli (APEC) and mammary pathogenic E. coli (MPEC). In certain embodiments, the ExPEC bacteria comprises APEC bacteria.

[0045] In embodiments, a composition induces an immune response to the antigen in a cell, tissue or animal (e.g., a human). As used herein, an "antigenic composition" (which alternatively may be referred to as an "immunogenic composition") may comprise an antigen (e.g., a protein, peptide, or polypeptide) or a modified version of an antigen. In particular embodiments the antigenic composition comprises or encodes all or part of the NhaA protein or a mutated version thereof, including a deglycosylated, or amino acid modified version thereof. In certain embodiments, the immunogenic composition or vaccine comprises at least one pharmaceutically acceptable carrier or adjuvant. In otherAgent Ref. P14443WO00 11 embodiments, the antigenic composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination. In certain embodiments, the antigenic composition is conjugated to or comprises an HLA anchor motif amino acids.

[0046] In certain embodiments, an antigenic composition or immunologically functional equivalent may be used as an effective vaccine in inducing an anti-ExPEC humoral and / or cell-mediated immune response in an animal, including human. The present disclosure contemplates one or more antigenic compositions or vaccines for use in both active and passive immunization embodiments.

[0047] A vaccine or immunogenic composition of the present disclosure may vary in its composition of proteinaceous components. It will be understood that various compositions described herein may further comprise additional components. For example, one or more vaccine or immunogenic composition components may be comprised in a lipid or liposome. In another non-limiting example, a vaccine or immunogenic composition may comprise one or more adjuvants. A vaccine or immunogenic composition of the present disclosure, and its various components, may be prepared and / or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.

[0048] It is understood that an immunogenic composition may be made by a method that is well known in the art, including but not limited to chemical synthesis by solid phase synthesis and purification away from the other products of the chemical reactions by HPLC, or production by the expression of a nucleic acid sequence (e.g., a DNA sequence) encoding a peptide or polypeptide comprising an antigen of the present disclosure in an in vitro translation system or in a living cell including, for example, in a yeast cell, bacterial, mammalian cells or baculovirus / insect cells. The antigenic composition may be isolated and extensively purified to remove one or more undesired small molecular weight molecules and / or lyophilized for more ready formulation into a desired vehicle. It is further understood that amino acid additions, deletions, mutations, chemical modification and such like that are made in an antigenic composition component, such as a vaccine, will preferably not substantially interfere with the antibody recognition of the epitopic sequence.

[0049] A peptide or polypeptide corresponding to one or more antigenic determinants of the receptor binding domain of the NhaA protein may generally be 10-20 amino acid residues in length, and may contain more than one peptide determinants or up to about 30-50 residues or so. A peptide sequence may be synthesized by methods known to those of ordinary skill in the art, such as, for example, peptide synthesis using automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, Calif.).Agent Ref. P14443WO00 12

[0050] Longer peptides or polypeptides also may be prepared, e.g., by recombinant means. In certain embodiments, a nucleic acid encoding an antigenic composition and / or a component described herein may be used, for example, to produce an antigenic composition in vitro or in vivo for the various compositions and methods of the present disclosure. For example, in certain embodiments, a nucleic acid encoding an antigen is comprised in, for example, a vector in a recombinant cell. The nucleic acid may be expressed to produce a peptide or polypeptide comprising an antigenic sequence. The peptide or polypeptide may be secreted from the cell, or comprised as part of or within the cell. A. Immunologically Functional Equivalents

[0051] As modifications and changes may be made in the structure of an antigenic composition of the present disclosure, and still obtain molecules having like or otherwise desirable characteristics, such immunologically functional equivalents are also encompassed within the present disclosure.

[0052] For example, certain amino acids may be substituted for other amino acids in a peptide, polypeptide or protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules or receptors, DNA binding sites, or such like. Since it is the interactive capacity and nature of a peptide, polypeptide or protein that defines its biological (e.g., immunological) functional activity, certain amino acid sequence substitutions can be made in an amino acid sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a peptide or polypeptide with like (agonistic) properties. It is thus contemplated by the inventors that various changes may be made in the sequence of an antigenic composition such as, for example a NhaA peptide or polypeptide without appreciable loss of biological utility or activity. In particular cases, there are one or more other amino acids that are modified compared to the corresponding wild-type sequence.

[0053] As used herein, an "amino molecule" refers to any amino acid, amino acid derivative or amino acid mimic as would be known to one of ordinary skill in the art. In certain embodiments, the residues of the antigenic composition comprises amino molecules that are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the antigenic composition may be interrupted by one or more non-amino molecule moieties.

[0054] Accordingly, antigenic compositions, particularly an immunologically functional equivalent of the sequences disclosed herein, may encompass an amino molecule sequence comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid.

[0055] In term of immunologically functional equivalent, it is well understood by the skilled artisan that, inherent in the definition is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable levelAgent Ref. P14443WO00 13 of equivalent immunological activity. An immunologically functional equivalent peptide or polypeptide are thus defined herein as those peptide(s) or polypeptide(s) in which certain, not most or all, of the amino acid(s) may be substituted.

[0056] In particular, where a shorter length peptide is concerned, it is contemplated that fewer amino acid substitutions should be made within the given peptide. A longer polypeptide may have an intermediate number of changes. The full length protein will have the most tolerance for a larger number of changes. Of course, a plurality of distinct polypeptides / peptides with different substitutions may easily be made and used in accordance with the disclosure.

[0057] It also is well understood that where certain residues are shown to be particularly important to the immunological or structural properties of a protein or peptide, e.g., residues in binding regions or active sites, such residues may not generally be exchanged. This is an important consideration in the present disclosure, where changes in the antigenic site should be carefully considered and subsequently tested to ensure maintenance of immunological function (e.g., antigenicity), where maintenance of immunological function is desired. In this manner, functional equivalents are defined herein as those peptides or polypeptides which maintain a substantial amount of their native immunological activity.

[0058] Amino acid substitutions are generally based on the relative similarity of the amino acid side- chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as immunologically functional equivalents.

[0059] To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (- 0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0060] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein, polypeptide or peptide is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within .+-.2 is preferred, those which are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.Agent Ref. P14443WO00 14

[0061] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the immunological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments, as in certain embodiments of the present disclosure. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a immunological property of the protein.

[0062] As detailed in U.S. Pat. No.4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0063] In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within .+-.2 is preferred, those which are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

[0064] Numerous scientific publications have also been devoted to the prediction of secondary structure, and to the identification of an epitope, from analyses of an amino acid sequence (Chou & Fasman, 1974a,b; 1978a,b, 1979). Any of these may be used, if desired, to supplement the teachings of U.S. Pat. No.4,554,101.

[0065] Moreover, computer programs are currently available to assist with predicting an antigenic portion and an epitopic core region of one or more proteins, polypeptides or peptides. Examples include those programs based upon the Jameson-Wolf analysis (Jameson & Wolf, 1988; Wolf et al., 1988), the program PepPlot™ (Brutlag et al., 1990; Weinberger et al., 1985), and other new programs for protein tertiary structure prediction (Fetrow & Bryant, 1993). Another commercially available software program capable of carrying out such analyses is MacVector (IBI, New Haven, Conn.).

[0066] In further embodiments, major antigenic determinants of a peptide or polypeptide may be identified by an empirical approach in which portions of a nucleic acid encoding a peptide or polypeptide are expressed in a recombinant host, and the resulting peptide(s) or polypeptide(s) tested for their ability to elicit an immune response. For example, PCR can be used to prepare a range of peptides or polypeptides lacking successively longer fragments of the C-terminus of the amino acid sequence. The immunoactivity of each of these peptides or polypeptides is determined to identify those fragments or domains that are immunodominant. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinant(s) of the peptide or polypeptide to be more precisely determined.Agent Ref. P14443WO00 15

[0067] Another method for determining a major antigenic determinant of a peptide or polypeptide is the SPOTs™ system (Genosys Biotechnologies, Inc., The Woodlands, Tex.). In this method, overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody. An antigenic determinant of the peptides or polypeptides which are initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive sequence.

[0068] Once one or more such analyses are completed, an antigenic composition, such as for example a peptide or a polypeptide is prepared that contain at least the essential features of one or more antigenic determinants. An antigenic composition is then employed in the generation of antisera against the composition, and preferably the antigenic determinant(s).

[0069] While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid. Nucleic acids encoding these antigenic compositions also can be constructed and inserted into one or more expression vectors by standard methods (Sambrook et al., 1987), for example, using PCR cloning methodology.

[0070] In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide or polypeptide structure or to interact specifically with, for example, an antibody. Such compounds, which may be termed peptidomimetics, may be used in the same manner as a peptide or polypeptide of the disclosure and hence are also immunologically functional equivalents.

[0071] Certain mimetics that mimic elements of protein secondary structure are described in Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orientate amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule. B. Antigen Mutagenesis

[0072] In particular embodiments, an antigenic composition is mutated for purposes such as, for example, enhancing its immunogenicity or producing or identifying a immunologically functional equivalent sequence. Methods of mutagenesis are well known to those of skill in the art (Sambrook et al., 1987).

[0073] As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template- dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in theAgent Ref. P14443WO00 16 concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No.4,237,224, specifically incorporated herein by reference in its entirety.

[0074] In a preferred embodiment, site directed mutagenesis is used. Site-specific mutagenesis is a technique useful in the preparation of an antigenic composition, through specific mutagenesis of the underlying DNA. In general, the technique of site-specific mutagenesis is well known in the art. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of a mutant through the use of specific oligonucleotide sequence(s) which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the position being mutated. Typically, a primer of about 17 to about 75 nucleotides in length is preferred, with about 10 to about 25 or more residues on both sides of the position being altered, while primers of about 17 to about 25 nucleotides in length being more preferred, with about 5 to 10 residues on both sides of the position being altered.

[0075] In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. As will be appreciated by one of ordinary skill in the art, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

[0076] This mutagenic primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as, for example, E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.Agent Ref. P14443WO00 17

[0077] Alternatively, a pair of primers may be annealed to two separate strands of a double stranded vector to simultaneously synthesize both corresponding complementary strands with the desired mutation(s) in a PCR reaction. A genetic selection scheme to enrich for clones incorporating the mutagenic oligonucleotide has been devised (Kunkel et al., 1987). Alternatively, the use of PCR with commercially available thermostable enzymes such as Taq polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector (Tomic et al., 1990; Upender et al., 1995). A PCR employing a thermostable ligase in addition to a thermostable polymerase also may be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector (Michael 1994).

[0078] The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

[0079] Additionally, one particularly useful mutagenesis technique is alanine scanning mutagenesis in which a number of residues are substituted individually with the amino acid alanine so that the effects of losing side-chain interactions can be determined, while minimizing the risk of large-scale perturbations in protein conformation (Cunningham et al., 1989). C. Liposome-Mediated Transfection

[0080] In a further embodiment of the disclosure, one or more vaccine or immunogenic composition components may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). D. Vaccine or Immunogenic Composition Component Purification

[0081] In any case, a vaccine component (e.g., an antigenic peptide or polypeptide) may be isolated and / or purified from the chemical synthesis reagents, cell or cellular components. In a method of producing the vaccine or immunogenic composition component, purification is accomplished by any appropriate technique that is described herein or well-known to those of skill in the art (e.g., Sambrook et al., 1987). There is no general requirement that an antigenic composition of the present disclosure or other vaccine component always be provided in their most purified state. Indeed, it is contemplated that less substantially purified vaccine or immunogenic composition component, which is nonethelessAgent Ref. P14443WO00 18 enriched in the desired compound, relative to the natural state, will have utility in certain embodiments, such as, for example, total recovery of protein product, or in maintaining the activity of an expressed protein. However, it is contemplated that inactive products also have utility in certain embodiments, such as, e.g., in determining antigenicity via antibody generation.

[0082] The present disclosure also provides purified, and in certain embodiments, substantially purified vaccines or immunogenic composition components. The term "purified vaccine component" or "purified immunogenic composition component" as used herein, is intended to refer to at least one respective vaccine or immunogenic composition component (e.g., a proteinaceous composition, isolatable from cells), wherein the component is purified to any degree relative to its naturally- obtainable state, e.g., relative to its purity within a cellular extract or reagents of chemical synthesis. In certain aspects wherein the vaccine component is a proteinaceous composition, a purified vaccine component also refers to a wild-type or mutant protein, polypeptide, or peptide free from the environment in which it naturally occurs.

[0083] Where the term "substantially purified" is used, this will refer to a composition in which the specific compound (e.g., a protein, polypeptide, or peptide) forms the major component of the composition, such as constituting about 50% of the compounds in the composition or more. In preferred embodiments, a substantially purified vaccine component will constitute more than about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or even more of the compounds in the composition.

[0084] In certain embodiments, a vaccine or immunogenic composition component may be purified to homogeneity. As applied to the present disclosure, "purified to homogeneity," means that the vaccine component has a level of purity where the compound is substantially free from other chemicals, biomolecules or cells. For example, a purified peptide, polypeptide or protein will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully. Various methods for quantifying the degree of purification of a vaccine component will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific protein activity of a fraction (e.g., antigenicity), or assessing the number of polypeptides within a fraction by gel electrophoresis.

[0085] Various techniques suitable for use in chemical, biomolecule or biological purification, well known to those of skill in the art, may be applicable to preparation of a vaccine component of the present disclosure. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; fractionation, chromatographic procedures, including but not limited to, partition chromatograph (e.g., paper chromatograph, thin- layer chromatograph (TLC), gas-liquid chromatography and gel chromatography) gas chromatography, high performance liquid chromatography, affinity chromatography, supercriticalAgent Ref. P14443WO00 19 flow chromatography ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity; isoelectric focusing and gel electrophoresis (see for example, Sambrook et al. 1989; and Freifelder, Physical Biochemistry, Second Edition, pages 238-246, incorporated herein by reference).

[0086] Given many DNA and proteins are known (see for example, the National Center for Biotechnology Information's GenBank™ and GenPept™ databases, or may be identified and amplified using the methods described herein, any purification method for recombinantly expressed nucleic acid or proteinaceous sequences known to those of skill in the art can now be employed. In certain aspects, a nucleic acid may be purified on polyacrylamide gels, and / or cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al.1989, incorporated herein by reference). In further aspects, a purification of a proteinaceous sequence may be conducted by recombinantly expressing the sequence as a fusion protein. Such purification methods are routine in the art. This is exemplified by the generation of an specific protein-glutathione S- transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose or the generation of a polyhistidine tag on the N- or C- terminus of the protein, and subsequent purification using Ni-affinity chromatography. In particular aspects, cells or other components of the vaccine may be purified by flow cytometry. Flow cytometry involves the separation of cells or other particles in a liquid sample, and is well known in the art (see, for example, U.S. Pat. Nos. 3,826,364, 4,284,412, 4,989,977, 4,498,766, 5,478,722, 4,857,451, 4,774,189, 4,767,206, 4,714,682, 5,160,974 and 4,661,913). Any of these techniques described herein, and combinations of these and any other techniques known to skilled artisans, may be used to purify and / or assay the purity of the various chemicals, proteinaceous compounds, nucleic acids, cellular materials and / or cells that may comprise a vaccine of the present disclosure. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified antigen or other vaccine component. E. Additional Vaccine Components

[0087] It is contemplated that an immunogenic composition of the disclosure may be combined with one or more additional components to form a more effective composition or vaccine. Non-limiting examples of additional components include, for example, one or more additional antigens, immunomodulators or adjuvants to stimulate an immune response to an antigenic composition of the present disclosure and / or the additional component(s). 1. Immunomodulators

[0088] For example, it is contemplated that immunomodulators can be included in the vaccine to augment a cell's or a patient's (e.g., an animal's) response. Immunomodulators can be included as purified proteins, nucleic acids encoding immunomodulators, and / or cells that expressAgent Ref. P14443WO00 20 immunomodulators in the vaccine composition, for example. The following sections list non-limiting examples of immunomodulators that are of interest, and it is contemplated that various combinations of immunomodulators may be used in certain embodiments (e.g., a cytokine and a chemokine).

[0089] Interleukins, cytokines, nucleic acids encoding interleukins or cytokines, and / or cells expressing such compounds are contemplated as possible vaccine components. Interleukins and cytokines, include but are not limited to interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-18, .beta.-interferon, .alpha.-interferon, .gamma.- interferon, angiostatin, thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, METH-1, METH-2, tumor necrosis factor, TGF.beta., LT and combinations thereof.

[0090] Chemokines, nucleic acids that encode for chemokines, and / or cells that express such also may be used as vaccine components. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine coding sequence in combination with, for example, a cytokine coding sequence, to enhance the recruitment of other immune system components to the site of treatment. Such chemokines include, for example, RANTES, MCAF, MIP1-alpha, MIP1-Beta, IP-10 and combinations thereof. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines.

[0091] In certain embodiments, an antigenic composition may be chemically coupled to a carrier or recombinantly expressed with a immunogenic carrier peptide or polypetide (e.g., a antigen-carrier fusion peptide or polypeptide) to enhance an immune reaction. Exemplary and preferred immunogenic carrier amino acid sequences include hepatitis B surface antigen, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as immunogenic carrier proteins. Means for conjugating a polypeptide or peptide to a immunogenic carrier protein are well known in the art and include, for example, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

[0092] It may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); low-dose cyclophosphamide (CYP; 300 mg / m.sup.2) (Johnson / Mead, NJ), or a gene encoding a protein involved in one or more immune helper functions, such as B-7. 2. Adjuvants

[0093] Immunization protocols have used adjuvants to stimulate responses for many years, and as such adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens areAgent Ref. P14443WO00 21 precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation. In one aspect, an adjuvant effect is achieved by use of an agent, such as alum, used in about 0.05 to about 0.1% solution in phosphate buffered saline. Alternatively, the antigen is made as an admixture with synthetic polymers of sugars (CarbopolTM) used as an about 0.25% solution. Adjuvant effect may also be made my aggregation of the antigen in the vaccine by heat treatment with temperatures ranging between about 70°to about 101°C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cell(s) such as C. parvum, an endotoxin or a lipopolysaccharide component of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles, such as mannide mono-oleate (Aracel A), or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DATM) used as a block substitute, also may be employed.

[0094] Some adjuvants, for example, certain organic molecules obtained from bacteria, act on the host rather than on the antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D- isoglutamine [MDP]), a bacterial peptidoglycan. The effects of MDP, as with most adjuvants, are not fully understood. MDP stimulates macrophages but also appears to stimulate B cells directly. The effects of adjuvants, therefore, are not antigen-specific. If they are administered together with a purified antigen, however, they can be used to selectively promote the response to the antigen.

[0095] Adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Pat. No. 4,877,611). In certain embodiments, hemocyanins and hemoerythrins may also be used in the disclosure. The use of hemocyanin from keyhole limpet (KLH) is preferred in certain embodiments, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed.

[0096] Various polysaccharide adjuvants may also be used. For example, the use of various pneumococcal polysaccharide adjuvants on the antibody responses of mice has been described (Yin et al., 1989). The doses that produce optimal responses, or that otherwise do not produce suppression, should be employed as indicated (Yin et al., 1989). Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.

[0097] Another group of adjuvants are the muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D- isoglutamine) group of bacterial peptidoglycans. Derivatives of muramyl dipeptide, such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, are also contemplated.

[0098] U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptide derivative of muramyl dipeptide which is described for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol. It is the to be effective in activating human monocytes and destroying tumor cells, but is non-toxic in generally high doses. The compounds of U.S. Pat. No. 4,950,645 and PCT Patent Application WO 91 / 16347, are contemplated for use with cellular carriers and other embodiments of the present disclosure.Agent Ref. P14443WO00 22

[0099] Another adjuvant contemplated for use in the present disclosure is BCG. BCG (bacillus Calmette-Guerin, an attenuated strain of Mycobacterium) and BCG-cell wall skeleton (CWS) may also be used as adjuvants in the disclosure, with or without trehalose dimycolate. Trehalose dimycolate may be used itself. Trehalose dimycolate administration has been shown to correlate with augmented resistance to influenza virus infection in mice (Azuma et al., 1988). Trehalose dimycolate may be prepared as described in U.S. Pat. No.4,579,945.

[0100] BCG is an important clinical tool because of its immunostimulatory properties. BCG acts to stimulate the reticulo-endothelial system, activates natural killer cells and increases proliferation of hematopoietic stem cells. Cell wall extracts of BCG have proven to have excellent immune adjuvant activity. Molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG (Jacobs et al., 1987; Snapper et al., 1988; Husson et al., 1990; Martin et al., 1990).

[0101] Live BCG is an effective and safe vaccine used worldwide to prevent tuberculosis. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly 2 billion immunizations, BCG has a long record of safe use in man (Luelmo, 1982; Lotte et al., 1984). It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and there is a worldwide distribution network with experience in BCG vaccination. An exemplary BCG vaccine is sold as TICE™ BCG (Organon Inc., West Orange, N.J.).

[0102] Amphipathic and surface active agents, e.g., saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of adjuvants for use with the immunogens of the present disclosure. Nonionic block copolymer surfactants (Rabinovich et al., 1994; Hunter et al., 1991) may also be employed. Oligonucleotides are another useful group of adjuvants (Yamamoto et al., 1988). Quil A and lentinen are other adjuvants that may be used in certain embodiments of the present disclosure.

[0103] One group of adjuvants preferred for use in the disclosure are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in mammals. Of course, the detoxified endotoxins may be combined with other adjuvants to prepare multi-adjuvant-incorporated cells. For example, combination of detoxified endotoxins with trehalose dimycolate is particularly contemplated, as described in U.S. Pat. No.4,435,386. Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Pat. No. 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins, is also envisioned to be useful, as described in U.S. Pat. No.4,520,019.Agent Ref. P14443WO00 23

[0104] In other embodiments, the present disclosure contemplates that a variety of adjuvants may be employed in the membranes of cells, resulting in an improved immunogenic composition. The only requirement is, generally, that the adjuvant be capable of incorporation into, physical association with, or conjugation to, the cell membrane of the cell in question. Those of skill in the art will know the different kinds of adjuvants that can be conjugated to cellular vaccines in accordance with this disclosure and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others. Certain adjuvants particularly contemplated for use are the teichoic acids from Gram-cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts may also be employed in connection with the disclosure (Takada et al., 1995a).

[0105] Various adjuvants, even those that are not commonly used in humans, may still be employed in animals, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells. The toxicity or other adverse effects that may result from either the adjuvant or the cells, e.g., as may occur using non-irradiated tumor cells, is irrelevant in such circumstances.

[0106] One group of adjuvants preferred for use in some embodiments of the present disclosure are those that can be encoded by a nucleic acid (e.g., DNA or RNA). It is contemplated that such adjuvants may be encoded in a nucleic acid (e.g., an expression vector) encoding the antigen, or in a separate vector or other construct. These nucleic acids encoding the adjuvants can be delivered directly, such as for example with lipids or liposomes. 3. Excipients, Salts and Auxiliary Substances

[0107] An antigenic composition of the present disclosure may be mixed with one or more additional components (e.g., excipients, salts, etc.) which are pharmaceutically acceptable and compatible with at least one active ingredient (e.g., antigen). Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and combinations thereof.

[0108] An antigenic composition of the present disclosure may be formulated into the vaccine as a neutral or salt form. A pharmaceutically-acceptable salt, includes the acid addition salts (formed with the free amino groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. A salt formed with a free carboxyl group also may be derived from an inorganic base such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and combinations thereof.

[0109] In addition, if desired, an antigentic composition may comprise minor amounts of one or more auxiliary substances such as for example wetting or emulsifying agents, pH buffering agents, etc. which enhance the effectiveness of the antigenic composition or vaccine.Agent Ref. P14443WO00 24 F. Vaccine and Immunogenic Composition Preparations

[0110] Once produced, synthesized and / or purified, an antigen or other vaccine component may be prepared as a vaccine or immunogenic composition for administration to an individual. The preparation of a vaccine is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251, 4,601,903, 4,599,231, 4,599,230, and 4,596,792, all incorporated herein by reference. Such methods may be used to prepare a vaccine comprising an antigenic composition comprising a particular NhaA as active ingredient(s), in light of the present disclosure. In particular embodiments, the compositions of the present disclosure are prepared to be pharmacologically acceptable vaccines.

[0111] Pharmaceutical vaccine or immunogenic compositions of the present disclosure comprise an effective amount of NhaA dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one NhaA will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0112] Examples of pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329, incorporated herein by reference). The immunogenic composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0113] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.Agent Ref. P14443WO00 25

[0114] The immunogenic composition may be formulated in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

[0115] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0116] In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present disclosure. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in preferred embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

[0117] In certain embodiments the immunogenic composition is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the disclosure, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.Agent Ref. P14443WO00 26

[0118] In certain preferred embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.

[0119] Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.

[0120] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and / or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

[0121] The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciatedAgent Ref. P14443WO00 27 that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng / mg protein.

[0122] In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof. G. Vaccine or Immunogenic Composition Administration

[0123] The manner of administration of a vaccine or immunogenic composition may be varied widely. Any of the conventional methods for administration of a vaccine or immunogenic composition are applicable. For example, a vaccine may be conventionally administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, rectally, nasally, topically, in eye drops, locally, using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0124] A vaccination or immunogenic composition delivery schedule and dosages may be varied on a patient by patient basis, taking into account, for example, factors such as the weight and age of the patient, the type of disease being treated, the severity of the disease condition, previous or concurrent therapeutic interventions, the manner of administration and the like, which can be readily determined by one of ordinary skill in the art.

[0125] A vaccine or immunogenic composition may be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. For example, the intramuscular route may be preferred in the case of toxins with short half lives in vivo. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host. Precise amounts of an active ingredient required to be administered depend on the judgment of the practitioner. In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein However, a suitable dosage range may be, for example, of the order of several hundred micrograms active ingredient per vaccination. In other non- limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5Agent Ref. P14443WO00 28 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per vaccination, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. A suitable regime for initial administration and booster administrations (e.g., innoculations) are also variable, but are typified by an initial administration followed by subsequent inoculation(s) or other administration(s).

[0126] In many instances, it will be desirable to have multiple administrations of the vaccine or immunogenic composition, usually not exceeding six vaccinations, for example, more usually not exceeding four vaccinations and in some cases one or more, usually at least about three vaccinations. The vaccinations may be at from two to twelve week intervals, more usually from three to five week intervals, although longer intervals are encompassed herein. Periodic boosters may be desirable to maintain protective levels of the antibodies.

[0127] The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays. Other immune assays can be performed and assays of protection from challenge with ExPEC can be performed, following immunization. H. Kits of the Disclosure

[0128] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, an ExPEC immunogenic composition may be comprised in a kit. In a non-limiting example, an immunogenic composition comprising an NhaA protein or a mutant ExPEC bacteria be comprised in a kit.

[0129] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present disclosureAgent Ref. P14443WO00 29 also will typically include a means for containing the composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0130] The component(s) of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may comprise a container means for containing a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0131] Irrespective of the number and / or type of containers, the kits of the disclosure may also comprise, and / or be packaged with, an instrument for assisting with the injection / administration and / or placement of the ultimate composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, and / or any such medically approved delivery vehicle. In some cases, there are one or more means to identify the presence of ExPEC in a sample from an individual.

[0132] In embodiments, compositions of the present disclosure comprise a mutant ExPEC bacteria comprising a disruption of the endogenous nhaA gene. Said disruption may comprise deletion or mutation of the gene. Disrupting the endogenous nhaA gene can result in partial or complete inactivation of the NhaA protein. Inactivation of the NhaA protein significantly attenuates the virulence of the ExPEC bacteria in the bloodstreams of both mammals and avian animals but does not affect mutant growth in vitro. This makes nhaA an ideal target for the development of broadly conserved vaccine antigens and novel antimicrobial drugs. Thus, immunogenic compositions comprising mutant ExPEC bacteria having a disrupted nhaA gene can provide live, attenuated bacteria which exhibit high immunogenicity while at the same time not producing dangerous pathogenic or lethal effects.

[0133] The immunogenic and vaccine compositions of this disclosure are not, however, restricted to any particular type or method of preparation. These include, but are not limited to, infectious DNA vaccines (i.e., using plasmids, vectors or other conventional carriers to directly inject DNA into pigs), live vaccines, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, genetically engineered vaccines, etc. These vaccines are prepared by standard methods known in the art.

[0134] In some embodiments, the disruption of the endogenous nhaA gene comprises deletion or knockout of the nhaA gene, resulting in complete inactivation of the NhaA protein. In certain embodiments, this leads to attenuation of the mutant ExPEC’s virulence as compared to wild-type ExPEC bacteria.

[0135] In certain embodiments, the NhaA protein comprises one or more amino acid substitutions or deletions so that the NhaA protein is not naturally occurring. In some embodiments, the disruption ofAgent Ref. P14443WO00 30 the endogenous nhaA gene or NhaA protein comprises at least one mutation to loop-2, loop-3, loop-4, loop-5, loop-6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein. The mutation can comprise a knockout mutation or site mutation. In some embodiments, mutations to the nhaA gene or NhaA protein comprise at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO: 2. The at least one mutation can comprise an amino acid substitution. In certain embodiments, the amino acid substitution comprises one or more of the following amino acid substitutions: an arginine at position 225 when referenced to SEQ ID NO: 2 (H225R), a valine at position 127 when referenced to SEQ ID NO: 2 (A127V), a cysteine at position 163 when referenced to SEQ ID NO: 2 (D163C), a cysteine at position 164 when referenced to SEQ ID NO: 2 (D164C), and / or a cysteine at position 300 when referenced to SEQ ID NO: 2 (K300C). Thus the at least one mutation can comprise an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO: 2. Substitution mutations at one or more of these sites results in attenuated virulence of the mutant ExPEC bacteria as compared to wild-type ExPEC bacteria.

[0136] In some embodiments, the present disclosure includes immunogenic compositions comprising a live, attenuated mutant ExPEC bacteria and a pharmaceutically acceptable carrier. As used herein, the expression “live, attenuated mutant ExPEC bacteria” encompasses any live, mutant ExPEC bacteria that includes one or more of the mutations and / or deletions described herein. The pharmaceutically acceptable carrier can be, e.g., water, diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, stabilizing agent, culture medium, buffer, and the like. The composition can further include more than one pharmaceutically acceptable carrier.

[0137] The term “adjuvant” refers to a compound that enhances the effectiveness of the immunogenic compositions and may be added to the formulation that includes the immunizing agent. Adjuvants provide enhanced immune response even after administration of only a single dose of the vaccine. Adjuvants may include, for example, aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), non-metabolizable oil, mineral and / or plant / vegetable and / or animal oils, polymers, carbomers, surfactants, natural organic compounds, plant extracts, carbohydrates, cholesterol, lipids, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, HRA-3 (acrylic acid saccharide cross-linked polymer), HRA-3 with cottonseed oil (CSO), or preferably an acrylic acid polyol cross-linked polymer. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopeia type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri-(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination withAgent Ref. P14443WO00 31 emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the PLURONIC® brand products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart- Tull, D. E. S.) John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997). In a preferred embodiment the adjuvant is at a concentration of about 0.01 to about 50 wt. %, preferably at a concentration of about 2 wt. % to 30 wt. %, more preferably at a concentration of about 5 wt. % to about 25 wt. %, still more preferably at a concentration of about 7 wt. % to about 22 wt. %, and most preferably at a concentration of about 10 wt. % to about 20 wt. % of the final product. Examples of suitable adjuvants are described in U.S. Patent Application Publication No. US2004 / 0213817 A1.

[0138] “Diluents”, as used herein, can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others.

[0139] Immunogenic compositions comprising the attenuated mutant ExPEC bacteria can be prepared in the form of a suspension or in a lyophilized form or, alternatively, in a frozen form. If frozen, glycerol or other similar agents may be added to enhance stability when frozen. The advantages of live attenuated bacteria, in general, include the presentation of all the relevant immunogenic determinants of an infectious agent in its natural form to the host's immune system, and the need for relatively small amounts of the immunizing agent due to the ability of the agent to multiply in the immunized host.

[0140] Attenuation of the bacteria for a live vaccine, so that it is insufficiently pathogenic to substantially harm the immunized subject, may be accomplished by known procedures, including chemical mutagenesis or serial passaging. In some embodiments, the live, attenuated mutant ExPEC is attenuated such that when the attenuated bacteria is administered to a subject it fails to cause clinical signs of ExPEC infection but is capable of inducing an immune response that immunizes the subject against pathogenic infection by ExPEC.

[0141] The immunogenic compositions of the present disclosure can be formulated following accepted convention to include acceptable carriers for animals or humans, such as standard buffers, stabilizers, diluents, preservatives, and / or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing.Agent Ref. P14443WO00 32

[0142] The immunogenic compositions of the present disclosure may further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others. Non- limiting examples of adjuvants that can be used in the vaccine of the present disclosure include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, ionic polysaccharides, and Avridine lipid-amine adjuvant. Non- limiting examples of oil-in-water emulsions useful in the vaccine of the disclosure include modified SEAM62 and SEAM 1 / 2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v / v) squalene (Sigma), 1% (v / v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v / v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v / v) ethanol, 200 μg / ml Quil A, 100 μg / ml cholesterol, and 0.5% (v / v) lecithin. Modified SEAM 1 / 2 is an oil-in-water emulsion comprising 5% (v / v) squalene, 1% (v / v) SPAN® 85 detergent, 0.7% (v / v) Tween 80 detergent, 2.5% (v / v) ethanol, 100 μg / ml Quil A, and 50 μg / ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines. Additional adjuvant systems permit for the combination of both T-helper and B-cell epitopes, resulting in one or more types of covalent T-B epitope linked structures, which may be additionally lipidated, such as those described in WO2006 / 084319, WO2004 / 014957, and WO2004 / 014956.

[0143] In some embodiments, the live, attenuated mutant ExPEC comprises a deletion or knockout of the nhaA gene. In some embodiments, the live, attenuated mutant ExPEC comprises at least one mutation to loop-2, loop-3, loop-4, loop-5, loop-6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein. In some embodiments, the live, attenuated mutant ExPEC comprises one or more of the following amino acid substitutions: an arginine at position 225 when referenced to SEQ ID NO: 2 (H225R), a valine at position 127 when referenced to SEQ ID NO: 2 (A127V), a cysteine at position 163 when referenced to SEQ ID NO: 2 (D163C), a cysteine at position 164 when referenced to SEQ ID NO: 2 (D164C), and / or a cysteine at position 300 when referenced to SEQ ID NO: 2 (K300C).

[0144] Additional genetically engineered vaccines may be produced by techniques known in the art. Such techniques involve, but are not limited to, further manipulation of recombinant DNA, modification of or substitutions to the amino acid sequences of the recombinant proteins and the like.

[0145] An immunologically effective amount of the immunogenic composition of the present disclosure is administered to a subject in need of protection against bacterial infection. The immunologically effective amount or the immunogenic amount that inoculates the subject can be easily determined or readily titrated by routine testing. An effective amount is one in which a sufficient immunological response to the immunogenic composition is attained to protect the subject exposed toAgent Ref. P14443WO00 33 ExPEC. Preferably, the subject is protected to an extent in which one to all of the adverse physiological symptoms or effects of the bacterial disease are significantly reduced, ameliorated or totally prevented.

[0146] The appropriate dose of the immunogenic composition of the present disclosure depends on several variables such as the formulation, the route of administration, the subject’s age, the animal's weight, the time of administration, the excretion rate, and reaction irritability. One of ordinary skill in the art can determine the appropriate dose by administering the antigen to the animal and assaying for an increase or, if applicable, a decrease in the immune response.

[0147] Immunogenic compositions of the present disclosure can be administered by any means that achieve the intended purpose, using a composition as described herein. For example, route of administration of such a composition can be by parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and intravenous administration. In one embodiment of the present disclosure, the composition is administered by intramuscularly. Parenteral administration can be by bolus injection or by gradual perfusion over time. Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan. Administration that is oral, or alternatively, subcutaneous, may be preferred. Oral administration may be direct, via water, or via feed (solid or liquid feed). When provided in liquid form, the vaccine may be lyophilized with reconstitution, or provided as a paste, for direct addition to feed (mix in or top dress) or otherwise added to water or liquid feed. How to Make Mutants

[0148] ExPEC bacteria having a mutation, i.e. a mutation in the nhaA gene encoding the NhaA protein, may be identified or created using standard techniques, for example, chemical induction or recombinant DNA technology or combinations thereof. One possible way of mutating a gene encoding the NhaA protein is by means of classical methods such as the treatment of ExPEC bacteria with mutagenic agents such as base analogues, treatment with ultraviolet light or temperature treatment (Anderson, P. 1995. Mutagenesis, p 3158 in Methods in Cell Biology 48. H. F. Epstein and D.C. Shakes (Eds)).

[0149] The exact nature of the mutation caused by classical mutation techniques is usually unknown. This can be a point mutation which may eventually revert to wild-type. In some cases, it may be desirable to make the mutant ExPEC bacteria using transposon mutagenesis or recombinant DNA techniques. Mutation by transposon mutagenesis is a mutagenesis-technique well-known in the art that can be used to create a mutation at a localized site in the chromosome.Agent Ref. P14443WO00 34

[0150] In some embodiments, a mutation is introduced at a predetermined site using recombinant DNA-technology. Recombinant DNA techniques relate to cloning of the gene, modification of the gene sequence by site-directed mutagenesis, restriction enzyme digestion followed by re-ligation or PCR-approaches and to subsequent replacement of the wild type gene with the mutant gene (allelic exchange or allelic replacement). Standard recombinant DNA techniques such as cloning the gene in a plasmid, digestion of the gene with a restriction enzyme, followed by endonuclease treatment, re- ligation and homologous recombination in the host strain, are all known in the art and described i.a. in Maniatis / Sambrook (Sambrook, J. et al. Molecular cloning: a laboratory manual. ISBN 0-87969-309- 6). Site-directed mutations can e.g. be made by means of in vitro site directed mutagenesis using the TRANSFORMER® kit sold by Clontech. PCR-techniques are extensively described in (Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN 0-87969-447-5 (1995).

[0151] A mutation may be introduced at a predetermined site in genomic DNA via an insertion, a deletion, or a substitution of one nucleotide by another, such as a point mutation with the only proviso that the mutated gene encodes no corresponding NhaA protein, a non-functional NhaA protein, or a reduced-function NhaA protein. The mutation should produce a bacteria with no NhaA protein, a non- functional NhaA protein, or a reduced-function NhaA protein. In some embodiments, the mutation is a deletion mutation, where disruption of the gene is caused by the excision of nucleic acids. Such a mutation can e.g. be made by deletion of a number of base pairs. Even very small deletions such as stretches of 10 base pairs can already cause the gene to encode no protein or a non-functional protein. Even the deletion of one single base pair may lead to no protein or a non-functional protein, since as a result of such a mutation, the other base pairs are no longer in the correct reading frame or transcription has been inhibited or diminished. In some embodiments, a longer stretch is removed e.g. 100 base pairs. In other embodiments, the whole gene is deleted. Well-defined and deliberately made mutations involving the deletion of fragments or the whole gene of a gene of the NhaA protein have the advantage, in comparison to classically induced mutations, that they will not revert to the wild-type situation. Antibodies

[0152] Also contemplated by the present disclosure are anti-NhaA antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized, human, avian, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize an NhaA polypeptide of the disclosure. The term “specific for” indicates that the variable regions of the antibodies of the disclosure recognize and bind an NhaA polypeptide exclusively (i.e., are able to distinguish a single NhaA polypeptide from related polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), and which are permitted (optionally) toAgent Ref. P14443WO00 35 interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the Ab molecule. Screening assays to determine binding specificity of an antibody of the disclosure are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the NhaA polypeptides of the disclosure are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, a NhaA polypeptide of the disclosure from which the fragment was derived.

[0153] For the purposes of clarity, “antibody” refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen. Immunoglobulins are serum proteins composed of “light” and “heavy” polypeptide chains having “constant” and “variable” regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions. Antibodies can exist in a variety of forms including, for example, as, Fv, Fab′, F(ab′)2, as well as in single chains, and include synthetic polypeptides that contain all or part of one or more antibody single chain polypeptide sequences. Methods of Inducing an Immune Response Against ExPEC

[0154] The present disclosure also provides methods of inducing an immune response against ExPEC in a subject. In certain embodiments, a method of inducing an immune response against ExPEC comprises administering to the subject an NhaA protein of an extraintestinal pathogenic Escherichia coli (ExPEC) bacteria; and a pharmaceutically acceptable carrier. In embodiments of the disclosure, a method of inducing an immune response against ExPEC in a subject comprises administering to the subject an immunogenic composition comprising a mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene according to the present disclosure. In some embodiments, the mutant ExPEC bacteris is an ExPEC variant, including, for example, uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), those isolates responsible for septicemia (SEPEC), avian pathogenic E. coli (APEC) and mammary pathogenic E. coli (MPEC). In certain embodiments, the ExPEC bacteria comprises APEC.

[0155] As appreciated by one skilled in the art, administration of the immunogenic composition can be effected by any suitable method, including but not limited to parenteral injection, intranasal administration, intrapharyngeal administration, subcutaneous injection, intramuscular injection, oral administration, or topical administration.

[0156] In embodiments of the disclosure, the subject can be a human or an animal that is need of protection against diseases, infections, and / or pathologies caused by infection with ExPEC. ForAgent Ref. P14443WO00 36 example, the subject may be an animal, including a human, avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, and porcine animal. Subjects may also be domesticated animals such as cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs, and the like. Avian animals includes poultry animals, such as chickens, turkeys, ducks, geese, guinea fowl, pigeons, ostrich, emu, partridge, pheasant, and the like. In certain embodiments, the subject is a mammal, such as a human. In certain embodiments, the subject is a poultry animal.

[0157] The determination of the dosage of the immunogenic composition to be administered is well within one skilled in the art. Typically, in the case of a live attenuated vaccine, the amount of the bacteria can be in a range of about 1 bacterium to about 10,000,000 bacteria per administration depending on the route administered, the particular subject in need of treatment, and the size and health of the subject. In certain embodiments, the dose is from about 1 bacterium to about 10,000,000 bacterium, from about 100 bacterium to about 1,000,000 bacterium, from about 1,000 bacterium to about 100,000 bacterium, or any range therein.

[0158] As appreciated by one skilled in the art, the number of doses and frequency of dosing will vary depending on the route administered, the particular subject in need of treatment, and the size and health of the subject. In some embodiments, the immunogenic composition is administered in a single dose. In other embodiments, the immunogenic composition is administered in two or more doses. Embodiments

[0159] Various embodiments of the compositions, systems, and methods described herein are set forth in the following set of numbered embodiments.

[0160] 1. An immunogenic composition comprising: an NhaA protein of an extraintestinal pathogenic Escherichia coli (ExPEC) bacteria; and a pharmaceutically acceptable carrier.

[0161] 2. The immunogenic composition of embodiment 1, wherein the ExPEC bacteria comprises avian pathogenic E. coli (APEC) bacteria.

[0162] 3. The immunogenic composition of embodiment 1 or 2, wherein the pharmaceutically acceptable carrier comprises a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combinations thereof.

[0163] 4. The immunogenic composition of any one of embodiments 1-3, wherein the NhaA protein comprises one or more amino acid substitutions or deletions so that said protein is not naturally occurring.

[0164] 5. The immunogenic composition of embodiment 4, wherein the one or more amino acid substitutions or deletions of the NhaA protein comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO: 2.Agent Ref. P14443WO00 37

[0165] 6. The immunogenic composition of embodiment 4 or 5, wherein the one or more amino acid substitutions or deletions of the NhaA protein comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO: 2.

[0166] 7. An immunogenic composition comprising: a mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene; and a pharmaceutically acceptable carrier.

[0167] 8. The immunogenic composition of embodiment 7, wherein the ExPEC bacteria comprises avian pathogenic E. coli (APEC) bacteria.

[0168] 9. The immunogenic composition of embodiment 7 or 8, wherein the disruption of the endogenous nhaA gene comprises deletion of the nhaA gene.

[0169] 10. The immunogenic composition of any one of embodiments 7-9, wherein the disruption of the endogenous nhaA gene results in complete inactivation of the NhaA protein.

[0170] 11. The immunogenic composition of any one of embodiments 7-10, wherein the disruption of the endogenous nhaA gene comprises at least one mutation to loop-2, loop-3, loop-4, loop-5, loop-6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein.

[0171] 12. The immunogenic composition of any one of embodiments 7-11, wherein the mutant ExPEC bacteria exhibits attenuated virulence as compared to wild-type ExPEC bacteria.

[0172] 13. The immunogenic composition of any one of embodiments 7-12, wherein the disruption of the endogenous nhaA gene comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO: 2.

[0173] 14. The immunogenic composition of embodiment 13, wherein the at least one mutation comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO: 2.

[0174] 15. The immunogenic composition of any one of embodiments 7-14, wherein the at least one mutation results in attenuated virulence of the mutant ExPEC bacteria as compared to wild-type ExPEC bacteria.

[0175] 16. The immunogenic composition of any one of embodiments 7-15, wherein the mutant ExPEC bacteria is a live, attenuated bacteria.

[0176] 17. The immunogenic composition of any one of embodiments 7-16, wherein the pharmaceutically acceptable carrier comprises a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combinations thereof.

[0177] 18. A method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject, comprising: administering to the subject an immunogenic composition comprising a mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene.Agent Ref. P14443WO00 38

[0178] 19. The method of embodiment 18, wherein the ExPEC bacteria comprises avian pathogenic E. coli (APEC) bacteria.

[0179] 20. The method of embodiment 18 or 19, wherein the disruption of the endogenous nhaA gene comprises deletion of the nhaA gene.

[0180] 21. The method of any one of embodiments 18-20, wherein the disruption of the endogenous nhaA gene results in complete inactivation of the NhaA protein.

[0181] 22. The method of any one of embodiments 18-21, wherein the disruption of the endogenous nhaA gene comprises at least one mutation to loop-2, loop-3, loop-4, loop-5, loop-6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein.

[0182] 23. The method of any one of embodiments 18-22, wherein the mutant ExPEC bacteria exhibits attenuated virulence as compared to wild-type ExPEC bacteria.

[0183] 24. The method of any one of embodiments 18-23, wherein the disruption of the endogenous nhaA gene comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO: 2.

[0184] 25. The method of embodiment 24, wherein the at least one mutation comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO: 2.

[0185] 26. The method of any one of embodiments 18-25, wherein the at least one mutation results in attenuated virulence of the mutant ExPEC bacteria as compared to wild-type ExPEC bacteria.

[0186] 27. The method of any one of embodiments 18-26, wherein the mutant ExPEC bacteria is a live, attenuated bacteria.

[0187] 28. The method of any one of embodiments 18-27, wherein the subject is a poultry animal, mammal, or human.

[0188] 29. The method of any one of embodiments 18-28, wherein administration of the mutant ExPEC bacteria immunizes the subject against a pathology caused by ExPEC bacteria.

[0189] 30. The method of any one of embodiments 18-29, further comprising pharmaceutically acceptable carrier.

[0190] 31. The method of claim any one of embodiments 18-30, wherein the pharmaceutically acceptable carrier comprises a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combinations thereof.

[0191] 32. The method of claim any one of embodiments 18-31, wherein the immunogenic composition is administered intranasally, intramuscularly, intradermally, subcutaneously, or orally.

[0192] 33. The method of any one of embodiments 18-32, wherein the immunogenic composition is administered in a single dose.Agent Ref. P14443WO00 39

[0193] 34. The method of any one of embodiments 18-33, wherein the immunogenic composition is administered in two or more doses.

[0194] 35. A method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject, comprising: administering to the subject an NhaA protein of an extraintestinal pathogenic Escherichia coli (ExPEC) bacteria; and a pharmaceutically acceptable carrier.

[0195] 36. The method of embodiment 35, wherein the NhaA protein comprises one or more amino acid substitutions or deletions so that said protein is not naturally occurring.

[0196] 37. The method of embodiment 35 or 36, wherein administration of the NhaA protein immunizes the subject against a pathology caused by ExPEC bacteria.

[0197] 38. The method of any one of embodiments 35-37, wherein the subject is a poultry animal, mammal, or human.

[0198] 39. The method of any one of embodiments 35-38, wherein the pharmaceutically acceptable carrier comprises a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combinations thereof.

[0199] 40. A mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene.

[0200] 41. The mutant ExPEC bacteria of embodiment 40, wherein the disruption of the endogenous nhaA gene results in complete inactivation of the NhaA protein.

[0201] 42. The mutant ExPEC bacteria of embodiment 40 or 41, wherein the disruption of the endogenous nhaA gene comprises deletion of the nhaA gene.

[0202] 43. The mutant ExPEC bacteria of any one of embodiments 40-42, wherein the disruption of the endogenous nhaA gene comprises at least one mutation to loop-2, loop-3, loop-4, loop-5, loop- 6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein.

[0203] 44. The mutant ExPEC bacteria of any one of embodiments 40-43, wherein the disruption of the endogenous nhaA gene comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO: 2.

[0204] 45. The mutant ExPEC bacteria of embodiment 44, wherein the at least one mutation comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO: 2.

[0205] 46. The mutant ExPEC bacteria of any one of embodiments 40-45, wherein the mutant ExPEC bacteria exhibits attenuated virulence as compared to wild-type ExPEC bacteria.Agent Ref. P14443WO00 40 EXAMPLES Example 1. nhaA is Involved in ExPEC Virulence

[0206] A transposon-directed insertion-site sequencing strategy was used to identify virulence- essential ExPEC genes. nhaA was identified as an essential gene for systemic infection of ExPEC in mammalian and avian models. Furthermore, the attenuation of the ∆nhaA mutant strain was confirmed by testing the wild-type (WT) and mutant strains individually in chick and mouse models. The wild- type strain killed all eight chicks in 2 days; however, no death was observed in the ∆nhaA mutant group after 7 days (P < 0.001) (FIG. 1A). Additionally, nhaA deletion significantly decreased the bacterial load in the blood (P < 0.001) (Fig.1B). An average of 1 × 108CFU / mL bacteria was identified in the blood from WT group chicks; however, no bacteria could be isolated from the blood samples of mutant group chicks (limit of detection, 50 CFU / mL blood). Furthermore, nhaA gene reintroduction into the mutants almost restored chick mortality and bacterial loads in serum to the WT levels. Similar results were observed in the mouse model. When their growth was compared in M9 minimal medium, ∆nhaA mutant and WT showed no significant differences in growth in vitro (FIG.1E).

[0207] These results demonstrate that nhaA is required for ExPEC virulence in chick and mouse models of systemic infection. Example 2. Identification of Protein Loops and Amino Acid Residues of NhaA Contributing to ExPEC Virulence

[0208] NhaA protein is predicted to have a putative secondary structure comprising 12 transmembrane segments connected by 11 hydrophilic loops. The full NhaA sequence is shown in Table 1 below.Agent Ref. P14443WO00 41 Table 1 Sequence SEQ ID NhaA (nucleotide)GTGAAACATCTGCATCGATTCTTTAGCAGTGATGSEQ ID NO: 1 CCTCGGGAGGCATTATTCTCATTATTGCCGCTGT ATTAGCGATGATTATGGCCAACAGCGGTGCAAC CAGTGGATGGTATCACGACTTTCTTGAGACGCCG GTTCAGCTCCGGGTTGGGACACTTGAGATCAAC AAGAACATGCTGCTATGGATCAATGACGCTCTG ATGGCGGTATTTTTCCTGTTGGTTGGTCTGGAAG TTAAACGCGAGCTGATGCAAGGTTCGCTGGCCA GTCTGCGCCAGGCGGCATTTCCTGTTATTGCCGC AATCGGCGGGATGATTGTCCCGGCATTGCTCTAT CTGGCTTTTAACTATGCCGATCCGATTACCCGCG AAGGCTGGGCAATCCCGGCGGCGACTGACATTG CCTTTGCACTTGGTGTGTTGGCGCTGTTGGGAAG TCGTGTTCCGTTAGCGCTGAAGATCTTTTTGATG GCTCTGGCTATTATCGACGATCTTGGGGCCATCA TTATCATCGCATTGTTCTACACTAATGACTTATC GATGGCCTCTCTTGGCGTCGCGGCTGTAGCAATT GCGGTACTCGTGGTATTGAATCTGTGTGGTGTAC GCCGCACGGGCGTTTATATTCTGGTTGGCGTGGT GCTGTGGACAGCGGTGTTGAAATCGGGGGTTCA CGCAACCCTGGCTGGCGTCATTGTCGGCTTCTTT ATTCCTTTGAAAGAGAAGCATGGGCGCTCTCCG GCTAAACGTCTGGAGCATGTTTTGCATCCATGGG TGGCGTATCTGATTTTGCCGCTGTTTGCATTTGCT AATGCTGGCGTTTCACTGCAAGGTGTCACGCTGG AAGGTTTGACCTCCATCCTGCCATTAGGGATCAT CGCTGGTTTGCTGATTGGCAAGCCACTGGGTATT AGTCTGTTCTGCTGGTTGGCGCTGCGTTTGAAAT TGGCACATCTGCCAGAGGGAACGACTTACCAGC AAATTATGGCGGTTGGTATCCTGTGCGGTATCGG TTTTACTATGTCTATCTTTATTGCCAGCCTGGCAT TTGGTAGCGTAGATCCAGAACTGATTAACTGGG CAAAATTAGGTATCCTTGTCGGTTCAATTTCTTC GGCGGTAATTGGATATAGCTGGTTACGCGTTCGT TTACGTCCATCAGTTTGA NhaA (Amino MKHLHRFFSSDASGGIILIIAAVLAMIMANSGATSG SEQ ID NO: 2 Acid) WYHDFLETPVQLRVGTLEINKNMLLWINDALMAV FFLLVGLEVKRELMQGSLASLRQAAFPVIAAIGGM IVPALLYLAFNYADPITREGWAIPAATDIAFALGVL ALLGSRVPLALKIFLMALAIIDDLGAIIIIALFYTNDL SMASLGVAAVAIAVLVVLNLCGVRRTGVYILVGV VLWTAVLKSGVHATLAGVIVGFFIPLKEKHGRSPA KRLEHVLHPWVAYLILPLFAFANAGVSLQGVTLEG LTSILPLGIIAGLLIGKPLGISLFCWLALRLKLAHLPE GTTYQQIMAVGILCGIGFTMSIFIASLAFGSVDPELI NWAKLGILVGSISSAVIGYSWLRVRLRPSV

[0209] The loops correlated with ExPEC virulence were investigated by constructing 11 loop-mutant strains (FIG. 2A) and evaluating their pathogenicity in a chick model. The WT and loop1-mutantAgent Ref. P14443WO00 42 strains (pnhaAL1) killed all eight chicks in 4 days, the loop7-mutant strain (pnhaAL7) killed half of the infected chicks in 7 days, and no death was observed in other loop-mutant strains (P < 0.001) (FIG. 2B). Similar results were observed in a mouse model (FIG. 2D). These results indicate that loops 2– 11 are involved in ExPEC XM virulence.

[0210] Previous studies have shown that several amino acid residues of NhaA are responsible for the functions of the Na+ / H+antiporter and pH sensing and response (FIG. 2A). Eleven site-mutation strains were constructed, and their virulence was compared with that of the WT. Site mutations of P129L, T132C, and D133C, previously shown to affect the transportation of Na+and Li+, did not attenuate E. coli virulence, and all chicks were killed within 3 days. Additionally, two amino acid residues (H225R and G338S) were previously identified as essential for E. coli's pH sensing and response. The site mutation of H225R significantly attenuated virulence (P < 0.001); however, the G338S site mutation strain was not significantly different from the WT strain. Similarly, in the two- site mutations of A127V and E252C, which are important for Na+and Li+transportation and pH sensing, only A127V led to significant virulence attenuation (P < 0.01). Additionally, site mutations of D163C, D164C, and K300C, which resulted in complete inactivation of NhaA in a previous study, significantly attenuated virulence (FIGS.2C and 2E). Example 3. nhaA Deletion Compromises Envelope Integrity

[0211] It was examined whether nhaA deletion affects bacterial envelope integrity by first testing ∆nhaA’s resistance to ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), and a high concentration of NaCl. ∆nhaA growth was not significantly different from that of the WT and ∆nhaA-complemented strains on Luria–Bertani (LB) plates. Additionally, nhaA deletion did not reduce E. coli’s resistance to 1 or 2 mM EDTA; all strains showed similar sensitivities to EDTA (FIG.3A). Furthermore, the ∆nhaA strain was significantly slower than the WT and ∆nhaA-complemented strains on LB with 2% SDS (FIG. 3A). A similar growth phenotype was observed in LB with 4% NaCl. Additionally, nhaA mutant growth was significantly reduced compared with that of the WT and ∆nhaA-complemented strains (FIGS. 3B and 3C). These results suggest that nhaA deletion might damage the bacterial envelope integrity. Furthermore, we performed a periplasmic ribonuclease (RNase) leakage assay to compare the capacity of ∆nhaA, WT, and ∆nhaA-complemented strains to retain periplasmic proteins. When grown on RNase test agar plates, RNase leakage from the periplasm forms pink halos around the bacterial colonies. ∆nhaA showed significantly more leakage of periplasmic RNase into the medium than the WT and ∆nhaA-complemented strains (FIG. 3D), suggesting that nhaA deletion compromises bacterial envelope integrity.Agent Ref. P14443WO00 43 Example 4. nhaA Gene Deletion Reduces ExPEC Resistance to Serum Bactericidal Effects

[0212] The molecular mechanism by which NhaA contributes to virulence was explored by comparing resistance to phagocytosis and persistence in RAW264.7 macrophage cells. No significant difference was observed between the WT, ΔnhaA mutant, and ∆nhaA-complemented strains.

[0213] Next, the ability of these strains to survive in avian and human serum was assessed. The WT, ΔnhaA mutant, and ∆nhaA-complemented strains were incubated in 90% avian serum at an initial inoculation dose of 5.0 × 106CFU / mL. The WT strain could still grow in the avian serum, and after 4 h incubation, the cell numbers of the WT strain were increased to 8.5 × 108CFU / mL. In contrast, nhaA deletion significantly reduced the resistance of ExPEC to serum-bactericidal effects, and the surviving cell numbers dramatically decreased to 4.6 × 103CFU / mL with approximately 184,000-fold changes between the WT and ∆nhaA strains (P < 0.001). However, nhaA reintroduction into the mutant strain completely recovered ExPEC's resistance to the WT level (FIG.4A). An even more obvious difference was observed when the WT, ΔnhaA, and ∆nha-complemented strains were tested in human serum. Although the WT strain could still propagate in human serum, nhaA deletion completely abolished ExPEC‘s survival capability (FIG.4B), and no live bacteria were detected after 4 h of incubation.

[0214] Surprisingly, only the nhaA mutant cells, but not the WT cells, exhibited morphological aberrations under serum stress. Approximately 100% of the mutant cells displayed filaments and curved shapes when cultured in avian serum; however, they maintained their rod shape when cultured under normal growth conditions (LB) (FIG.4C). Moreover, under a transmission electron microscope, approximately 56% ∆nhaA cells had increased intermembrane space, invagination of the IM, and formation of intracellular vacuoles, compared with the WT and ∆nhaA-complementation strains (FIG. 4D). This indicates that nhaA gene deletion reduces ExPEC resistance to serum bactericidal effects Example 5. NhaA Contributes to the Resistance to Complement-Mediated Bacterial Killing

[0215] The complement system is a protein network in human and animal serums that directly kills bacteria. The WT, ΔnhaA, and ∆nhaA-complemented strains were tested to determine their resistance to complement-mediatedby incubation in avian serum with complement or inactivated complement. After 4 h incubation, WT and ∆nhaA-complemented strains proliferated from 5 × 106CFU / mL to approximately 1 × 109CFU / mL in either complement-active or inactivated avian sera. Moreover, the ΔnhaA mutant strain decreased from 5.0 × 106CFU / mL to < 1 × 104CFU / mL in the complement-active avian serum. However, complement inactivation recovered the mutant strain’s growth nearly to the WT level, suggesting that NhaA contributed to complement-mediated bacterial killing in avian serum (FIG. 5A). When tested in human complement-active or inactivated sera, the mutant strain could not survive in the complement-active serum. However, complement inactivationAgent Ref. P14443WO00 44 significantly enhanced the mutant’s survival in human serum; however, its growth capability was not restored to the WT level (FIG.5B). Example 6. NhaA Impairs C3b and MAC Depositions Through Complement Classical and Alternative Pathways

[0216] The complement system can be activated by three distinct pathways: classical (CP), lectin (LP), and alternative pathways (AP). The roles of these three complement pathways in killing WT and ∆nhaA strains were investigated. The strains were independently incubated in 50% normal human serum (NHS) and 50% NHS with blocked CP (C1q-depleted serum), AP (factor B-depleted serum), or LP (mannose-treated serum). No significant difference was observed in the survival of WT and ∆nhaA strains when incubated in CP- or AP-blocked sera; however, ∆nhaA survival in NHS (P < 0.05) and the LP-blocked serum was significantly less than that of the WT strain (P < 0.01). These results suggest that NhaA is involved in the resistance to CP and AP of complement-mediated bactericidal activity.

[0217] Activation of these three pathways leads to the production and deposition of the complement protein C3b and MACs on the pathogen surface. After incubation in 50% NHS for 2 h, C3b and MAC were deposited on the WT and ∆nhaA cells (FIGS. 6B and 6C), confirming that the complement system was involved in the death of both strains. Furthermore, C3b and MAC depositions on ∆nhaA cells increased significantly but decreased gradually on WT cells with time. After 2 h incubation, C3b and MAC depositions on ∆nhaA were 7.8 (P < 0.001) and 16.8 times (P < 0.01) higher than those on the WT strain, respectively. These results indicate NhaA impaired C3b and MAC depositions on WT cells. Example 7. NhaA Plays an Important Role in the Resistance to Serum Bactericidal Activity and APEC's Virulence

[0218] Several membrane proteins, OmpA, Prc, Iss, and NlpI, contribute to ExPEC resistance to serum-mediated bacterial killing. Their mutant strains were constructed, and their resistance to serum bactericidal effects was evaluated using an ex-vivo assay. All the WT and mutant strains were incubated in 90% avian serum at an initial inoculation dose of 5.0×106CFU / mL. After 4 h incubation, the WT, ∆iss, ∆ompA, and ∆nlpI mutant strains rapidly increased to approximately 1.0 × 109CFU / mL, suggesting that these genes (iss, ompA, or nlpI) did not contribute to ExPEC’s resistance to serum’s bactericidal effects. In contrast, the ∆nhaA mutant strain was < 4.9×103CFU / mL, 350,000-fold (P < 0.01) less than that of the WT (FIG.7A), suggesting that the gene nhaA was essential in the resistance to serum’s bactericidal effects. Furthermore, the mutant strain ∆prc proliferated in avian serum; however, at a significantly lower level than that of the WT, ∆iss, ∆ompA, and ∆nlpI mutant strains, suggesting that the gene prc was less vital compared with nhaA.Agent Ref. P14443WO00 45

[0219] Next, their pathogenicity was assessed using a chick model. As expected, all chicks in the WT, ∆ompA, and ∆iss groups died within 3 days. Additionally, six chicks from the ∆nlpI mutant group (75%) and two from the ∆prc mutant group (25%) died. In contrast, all chicks from the ∆nhaA mutant group survived, suggesting that nhaA deletion significantly attenuated the virulence (FIG. 7B). Overall, these in vivo and in vitro results demonstrate that the nhaA gene in IM is essential in the resistance to host serum’s bactericidal effects and virulence. Example 8. nhaA is Important for the Full Virulence of Other ExPEC Prototype Strains

[0220] Since the amino acid sequences of NhaA are conserved in all ExPEC strains, including human strains, the virulence contribution of NhaA in the neonatal meningitis-associated E. coli prototype strain RS218 and the uropathogenic prototype strains UTI89 and CFT073 was assessed. The resistance of nhaA mutant strains to serum’s bactericidal effect was examined in 50% human serum, and their virulence was examined in a mouse model of bacteremia since neonatal meningitis-associated and uropathogenic E. coli could cause bacteremia. nhaA gene deletion in CFT073, RS218, and UTI89 resulted in a 2,400- (P < 0.01), 412.5- (P < 0.001), and 29.6-fold (P < 0.01) reduction of E. coli level in human serum, respectively, compared with their WT strains (FIG. 8A). Furthermore, when tested in a mouse model of bacteremia, seven of the eight mice challenged with UTI89 died, and six of eight mice died when challenged with WT strains of RS218 and CFT073. No death was observed in the mice challenged with ∆nhaA mutant strains of RS218 (P < 0.01) or UTI89 (P < 0.001), and only one mouse died in the group challenged with CFT073 nhaA mutant strain (P < 0.05). These results suggest that NhaA is essential for ExPEC’s virulence. Discussion

[0221] ExPEC strains cause various infections, including human urinary tract and bloodstream infections and avian colibacillosis. Notably, antibiotic-resistant E. coli, particularly ExPEC, causes the most deaths among common bacterial pathogens. The ability to resist serum bactericidal activity and survive in the bloodstream is an essential virulence trait of the ExPEC strain. The above examples demonstrate that independent of its Na+ / H+antiporter activities, NhaA maintains intact cell envelope and morphology and contributes greatly to ExPEC’s serum resistance by facilitating the evasion of complement killing. Furthermore, it is demonstrated that nhaA deletion significantly attenuates the virulence of avian and human ExPECs. Therefore, the examples identify an important virulence- associated factor and describe its underlying virulence mechanisms.

[0222] The RNase leakage assay suggests that the OM of the ΔnhaA mutant shows increased permeability. Similarly, the SDS tolerance assay indicates that the integrity of the cell envelope of ΔnhaA is disrupted. Furthermore, damaged OM could enhance complement deposition and death ofAgent Ref. P14443WO00 46 the prc mutant of ExPEC strain RS218, consistent with the finding that ΔnhaA mutant strains carry more complements than the wild-type (WT) strain (FIG.6). The complement system is first activated in the bacterial OM, leading to bactericidal activity. Notably, the disrupted OM of ΔnhaA may increase the accessibility of bacterial binding targets of C1q and other components of the complement system; however, intact OM in the WT strain may efficiently block or interfere with docking and accessibility. Moreover, the bactericidal effect mediated by MAC initially occurs through damage to the OM, promoting its permeability and stimulating lethal alterations in the intramembrane (IM).

[0223] Additionally, NhaA may contribute to the homeostasis of IM where it is located. Therefore, nhaA absence can cause instability of the IM, increasing susceptibility to MAC attacks. Moreover, it was observed that complement inactivation can not recover ΔnhaA’s growth capability in human sera to the WT level (FIG.5).

[0224] Furthermore, upon entering the stationary phase, the increase in the OD600of the ΔnhaA mutant was significantly slower than that of the WT, and the mutant failed to achieve the high OD600 level of the WT eventually (FIG. 3B). As the stationary phase represents various stresses, such as nutrient limitation and increased acidity, these results suggest that ΔnhaA may have a disadvantage in dealing with stressful conditions. Additionally, the ΔnhaA mutant forms long filaments in the serum, and the WT maintains the normal rod shape. Notably, bacterial cell division and septation may be affected by different stresses, thereby forming long filamentous cells. Previous studies have shown that the filamentous UPEC phenotype can be activated in response to host innate immunity and results from adaptive strategies and stress responses. Moreover, as the cell elongates, more complements are attached, making the cells more susceptible. Therefore, the filamentous formation may explain why nhaA mutant cells recruit more C3b and MAC attacks than WT cells.

[0225] A few previously reported membrane factors (Iss, Prc, OmpA and NlpI), which are essential for virulence, were incorporated and their roles in serum resistance and virulence were compared with NhaA. Surprisingly, NhaA, Prc and NlpI, contributed to virulence in the animal model, and NhaA and Prc were involved in serum resistance (FIG.7). The inability to replicate the roles of these factors may be due to the presence of redundant alleles or different genetic backgrounds in different strains. This finding underlines the importance of strain differences when studying the contributions of virulence factors. Notably, among these factors, NhaA made the greatest contribution, and nhaA deletion resulted in 0% mortality, thereby fully protecting the animals from ExPEC infection. Next, NhaA contributed greatly to virulence in three other ExPEC prototype strains, CFT073 (B2 phylogenetic group, serotype O6: K2: H1), RS218 (B2 phylogenetic group, serotype O18: K1: H7), and UTI89 (B2 phylogenetic group, serotype O18: K1: H7) (FIG. 8). Given its conservativeness and role in ExPEC, NhaA is an ideal drug target, and a nhaA null mutant is a preferred candidate for live attenuated bacterial vaccines.Agent Ref. P14443WO00 47 Materials and Methods

[0226] Bacterial strains and culture conditions. ExPEC XM (O2: K1: H7), belonging to the phylogenetic E. coli reference (ECOR) B2, was isolated from the brain of a duck with septicemia and neurological symptoms. The bacterial strains (ExPEC XM, RS218, CFT073, UTI89, DH5α and their derivatives) were routinely cultured at 37℃ in LB medium. For growth studies, the bacteria were grown in M9 minimal salts supplemented with 2 mM MgSO4, 0.1 mM CaCl2, and glucose (0.5% v / v) added as an energy substrate. Selective antibiotics were added when necessary at the following concentrations: ampicillin, 100 μg / mL; chloramphenicol, 25 μg / mL.

[0227] Human and chick serums. Normal human serum (NHS), C1q-depleted, and factor B-depleted sera were purchased from Complement Technology, Inc. (Tyler, USA). C1q-depleted and factor B- depleted sera supplemented with 5 mM CaCl2 and 2 mM MgCl2 served as CP- and AP-blocked sera, respectively. Additionally, to block the LP pathway, NHS was treated with 100 mM mannose. For normal avian serum (NAS) isolation, fresh chick blood purchased from Harbin Veterinary Research Institute (Harbin, China) was collected in 50 mL tubes and incubated at 37℃ for 2 h. Next, the blood was incubated at 4℃ for 4 h. Afterward, the tubes were centrifuged at 1000 × g for 10 min at 4℃. Subsequently, the supernatants were collected, aliquoted, and kept at -80℃ until further use. Heat- inactive human serum (HIHS) and heat-inactive avian serum (HIAS) were prepared by heating the NHS and NAS at 56℃ for 30 min.

[0228] Recombinant DNA techniques. PCR, DNA ligation, electroporation, and DNA gel electrophoresis were performed. The DNA-modifying and restriction enzymes used were purchased from New England Biolabs (Ipswich, USA) or Thermo Fisher Scientific (Waltham, MA, USA). Moreover, restriction fragments, PCR products, and recombinant plasmids were purified using the MiniBEST DNA Fragment Purification or MinElute Gel Extraction Kit (Takara, Dalian, China) as recommended by the supplier.

[0229] Furthermore, deletion mutants of ExPEC XM, RS218, CFT073, and UTI89 were constructed using the bacteriophage lambda-red homologous recombination system described by Datsenko and Wanner. For complementation, the coding sequence of nhaA and its putative promoter regions were amplified from the XM strain and independently cloned into pGEN-MCS using EcoRI and BamHI restriction sites. Next, site-directed and loop mutations were constructed using the complementation plasmid. Primers used for mutagenesis and complementation are listed in Table 2. Table 2. Oligonucleotide sequences used as PCR primers. Primers Sequence (5'-3') SEQ ID General PCR for cloning pGEN-MCS- CCGGAATTCTGTCAAAGAGCGCGGTGTGG SEQ ID NO: 3 nhaA-FAgent Ref. P14443WO00 48 pGEN-MCS- CGCGGATCCTCGTCCTGTCAAACTGATGG SEQ ID NO: 4 nhaA-R For DeletionaDel-nhaA-F CGATGATTCGTGCGGGGTAAAATCGTGAAAACGA SEQ ID NO: 5 TCTATTCACCTGAAGAGAAATAAAAAGTgtaggctgga gctgcttcga Del-nhaA-R TTTCTCTCCCTGATAACAATGAAAAGGGAGCCGTT SEQ ID NO: 6 TATGGCTCCCCAGTACATCGTCCTGcatatgaatatcctcctt ag Del-iss-F ACTATCGTTTAATTGTTGTCACATAGGATTCTGCC SEQ ID NO: 7 GTTTTTAACAATGCAGGATAATAAGgtgtaggctggagctg cttc Del-iss-R AGAAGTATATTAATGAGCAGTGCAGATAGAGCTG SEQ ID NO: 8 CCCATATCGATGGGCAACTCATGCAAcatatgaatatcctc cttag Del-prc-F GTATGTCTTTGATTGTGCGCGCAGAACACCTGGTG SEQ ID NO: 9 TTCTGAAACGGAGGCCGGGCCAGGCgtgtaggctggagct gcttc Del-prc-R GATTTACGGCATCTTGCCGCTGTTAAAAAATCAGG SEQ ID NO: 10 CACAATTTCTTGTGCCTGATTGATAcatatgaatatcctcctt ag Del-ompA-F ACCGTGTTATCTCGTTGGAGATATTCATGGCGTAT SEQ ID NO: 11 TTTGGATGATAACGAGGCGCAAAAAgtgtaggctggagct gcttc Del-ompA-R AATTTACTAAAGGCGAAAAAAAAACCCCGCAGCT SEQ ID NO: 12 GCGGGGTTTTTCTACCAGACGAGAACcatatgaatatcctc cttag Del-nlpI-F ACGCCTTGTTAGCAACCGGGAACAGGACGTTCATT SEQ ID NO: 13 CAACCGTGGTCTTCGGGAgtgggaagtgtaggctggagctgcttc Del-nlpI-R CGCCCTCACCCGTTAAGGTGATGGCAATCAAAAA SEQ ID NO: 14 AGATTACGGGCTGATGTGTACGTCAGcatatgaatatcctc cttag For NhaA mutation ΔnhaA-D65C-F ACATGCTGCTATGGATCAATTGTGCTCTGATGGCG SEQ ID NO: 15 GTATTTTT ΔnhaA-D65C- AAAAATACCGCCATCAGAGCACAATTGATCCATA SEQ ID NO: 16 R GCAGCATGT ΔnhaA-A127V- CGATTACCCGCGAAGGCTGGGTTATCCCGGCGGC SEQ ID NO: 17 F GACTGACAT ΔnhaA-A127V- ATGTCAGTCGCCGCCGGGATAACCCAGCCTTCGCG SEQ ID NO: 18 R GGTAATCG ΔnhaA-P129L- CCCGCGAAGGCTGGGCAATCTTAGCGGCGACTGA SEQ ID NO: 19 F CATTGCCTT ΔnhaA-P129L- AAGGCAATGTCAGTCGCCGCTAAGATTGCCCAGC SEQ ID NO: 20 R CTTCGCGGG ΔnhaA-T132C- GCTGGGCAATCCCGGCGGCGTGTGACATTGCCTTT SEQ ID NO: 21 F GCACTTGG ΔnhaA-T132C- CCAAGTGCAAAGGCAATGTCACACGCCGCCGGGA SEQ ID NO: 22 R TTGCCCAGC ΔnhaA-D133C- GGGCAATCCCGGCGGCGACTTGTATTGCCTTTGCA SEQ ID NO: 23 F CTTGGTGT ΔnhaA-D133C- ACACCAAGTGCAAAGGCAATACAAGTCGCCGCCG SEQ ID NO: 24 R GGATTGCCCAgent Ref. P14443WO00 49 ΔnhaA-D163C- TTGATGGCTCTGGCTATTATCTGTGATCTTGGGGC SEQ ID NO: 25 F CATCATTA ΔnhaA-D163C- TAATGATGGCCCCAAGATCACAGATAATAGCCAG SEQ ID NO: 26 R AGCCATCAA ΔnhaA-D164C- ATGGCTCTGGCTATTATCGACTGTCTTGGGGCCAT SEQ ID NO: 27 F CATTATCA ΔnhaA-D164C- TGATAATGATGGCCCCAAGACAGTCGATAATAGC SEQ ID NO: 28 R CAGAGCCAT ΔnhaA-H225R- CGGTGTTGAAATCGGGGGTTAGGGCAACCCTGGC SEQ ID NO: 29 F TGGCGTCAT ΔnhaA-H225R- ATGACGCCAGCCAGGGTTGCCCTAACCCCCGATTT SEQ ID NO: 30 R CAACACCG ΔnhaA-E252C- GCTCTCCGGCTAAACGTCTGTGTCATGTTTTGCAT SEQ ID NO: 31 F CCATGGGT ΔnhaA-E252C- ACCCATGGATGCAAAACATGACACAGACGTTTAG SEQ ID NO: 32 R CCGGAGAGC ΔnhaA-K300C- TCGCTGGTTTGCTGATTGGCTGTCCACTGGGTATT SEQ ID NO: 33 F AGTCTGTT ΔnhaA-K300C- AACAGACTAATACCCAGTGGACAGCCAATCAGCA SEQ ID NO: 34 R AACCAGCGA ΔnhaA-G338S- TTGGTATCCTGTGCGGTATCTCCTTTACTATGTCTA SEQ ID NO: 35 F TCTTTAT ΔnhaA-G338S- ATAAAGATAGACATAGTAAAGGAGATACCGCACA SEQ ID NO: 36 R GGATACCAA ΔnhaA-Loop1- TGGCCAACAGCGGTGCAACCAACATGCTGCTATG SEQ ID NO: 37 F GATCAA ΔnhaA-Loop1- TTGATCCATAGCAGCATGTTGGTTGCACCGCTGTT SEQ ID NO: 38 R GGCCA ΔnhaA-Loop2- TTTTCCTGTTGGTTGGTCTGTTTCCTGTTATTGCCG SEQ ID NO: 39 F CAAT ΔnhaA-Loop2- ATTGCGGCAATAACAGGAAACAGACCAACCAACA SEQ ID NO: 40 R GGAAAA ΔnhaA-Loop3- CATTGCTCTATCTGGCTTTTGGCTGGGCAATCCCG SEQ ID NO: 41 F GCGGC ΔnhaA-Loop3- GCCGCCGGGATTGCCCAGCCAAAAGCCAGATAGA SEQ ID NO: 42 R GCAATG ΔnhaA-Loop4- TTGGTGTGTTGGCGCTGTTGTTTTTGATGGCTCTGG SEQ ID NO: 43 F CTAT ΔnhaA-Loop4- ATAGCCAGAGCCATCAAAAACAACAGCGCCAACA SEQ ID NO: 44 R CACCAA ΔnhaA-Loop5- TCATTATCATCGCATTGTTCATGGCCTCTCTTGGCG SEQ ID NO: 45 F TCGC ΔnhaA-Loop5- GCGACGCCAAGAGAGGCCATGAACAATGCGATGA SEQ ID NO: 46 R TAATGA ΔnhaA-Loop6- TCGTGGTATTGAATCTGTGTACGGGCGTTTATATT SEQ ID NO: 47 F CTGGT ΔnhaA-Loop6- ACCAGAATATAAACGCCCGTACACAGATTCAATA SEQ ID NO: 48 R CCACGA ΔnhaA-Loop7- TGCTGTGGACAGCGGTGTTGGCAACCCTGGCTGGC SEQ ID NO: 49 F GTCAT ΔnhaA-Loop7- ATGACGCCAGCCAGGGTTGCCAACACCGCTGTCC SEQ ID NO: 50 R ACAGCA ΔnhaA-Loop8- TCGGCTTCTTTATTCCTTTGCCATGGGTGGCGTATC SEQ ID NO: 51 F TGATAgent Ref. P14443WO00 50 ΔnhaA-Loop8- ATCAGATACGCCACCCATGGCAAAGGAATAAAGA SEQ ID NO: 52 R AGCCGA ΔnhaA-Loop9- CTAATGCTGGCGTTTCACTGATCATCGCTGGTTTG SEQ ID NO: 53 F CTGAT ΔnhaA-Loop9- ATCAGCAAACCAGCGATGATCAGTGAAACGCCAG SEQ ID NO: 54 R CATTAG ΔnhaA- GTCTGTTCTGCTGGTTGGCGATTATGGCGGTTGGT SEQ ID NO: 55 Loop10-F ATCCT ΔnhaA- AGGATACCAACCGCCATAATCGCCAACCAGCAGA SEQ ID NO: 56 Loop10-R ACAGAC ΔnhaA- TGTCTATCTTTATTGCCAGCGCAAAATTAGGTATC SEQ ID NO: 57 Loop11-F CTTGT ΔnhaA- ACAAGGATACCTAATTTTGCGCTGGCAATAAAGAT SEQ ID NO: 58 Loop11-R AGACA Underlined are restriction cutting sites; Capital letters represent homologous fragments of the deleted genes.

[0230] Animal experiments. Healthy 7-day-old specific pathogen-free white leghorn chicks and 6- week-old BALB / c mice that tested negative for antibodies to ExPEC were used in this study. The mice and chicks were purchased from WeiTong LiHua Experimental Animal Technology (Beijing, China) and the Harbin Veterinary Research Institute (Harbin, China), respectively. For virulence experiments in vivo, the chicks (n = 8) were injected with 5 × 107CFU of ExPEC XM and its derivatives intratracheally. To determine the bacterial load, chicks were euthanized after 12 h, and blood was serially diluted in sterile phosphate-buffered saline (PBS) and plated on LB agar. Next, the challenged chicks were observed for 7 days, and death or survival was recorded. For loop-mutant and site-mutant strains, the mice (n = 8) were injected with 5 × 107CFU of ExPEC XM and its derivatives intraperitoneally, and the bacterial load in blood and mortality were determined after 12 h and 24 h, respectively. For other ExPEC strains, challenge doses in RS218, CFT073 and their mutants were 5 × 107CFU, and UTI89 and its mutants were 5 × 106CFU. The challenged mice were observed for 7 days, and death or survival was recorded.

[0231] Envelope stress assays. The growth of the XM, ∆nhaA mutant, and ∆nhaA- complemented strains were recorded in advance. For the high EDTA and SDS resistance assays, LB-grown bacteria (OD600≈ 0.4) were washed thrice with double distilled water, and a 10-fold serial dilution was performed. Next, 10 μL of each dilution was spotted onto LB plates and LB plates supplemented with 1 mM EDTA, 2 mM EDTA, or 2% SDS. Next, the plates were incubated for 36 h at 37°C. For the high osmotic pressure resistance assay, the bacteria were grown in LB medium or LB medium containing 4% NaCl and incubated at 37°C with shaking at 180 rpm. Afterward, the optical density was measured every hour at 600 nm to assess bacterial growth. For the RNase leakage assay, the bacterial suspension from a single fresh colony was adjusted to OD600≈ 0.4 with double distilled water. Next, 10 μL of the culture was spotted onto LB agar plates containing 0.2% (w / w) yeast RNA and 2.5% (w / w) toluidine blue O and incubated for 24 h at 37°C.Agent Ref. P14443WO00 51

[0232] Cell culture and infection. RAW264.7 (available from ATCC) murine macrophage cells were cultured in Dulbecco's minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and incubated at 37°C in a 5% CO2atmosphere. For quantitative mass proteomics analysis, RAW264.7 cells (2.5 × 106cells / well) were seeded onto 12-well tissue culture plates and inoculated with bacterial culture diluted in DMEM (5% FBS) at a multiplicity of infection of 100:1. After centrifugation at 500 × g for 5 min at 25°C; the plates were incubated for 1 h at 37°C in 5% CO2. The infected cells were washed once in PBS and incubated in DMEM containing 200 μg / mL gentamicin at 37°C in 5% CO2 for the indicated times. Subsequently, three washes with PBS were performed, and the cells were lysed with 1 mL water. Serial dilutions of the antibiotic on LB were used to determine the intracellular CFU.

[0233] Serum survival assay. Overnight bacterial cultures in LB were diluted 1:100 and grown to an OD600of 0.4–0.6 at 37℃ with shaking at 180 rpm. Next, the strains were washed twice with PBS, and the OD600 values were normalized to 1.0 (5 × 108CFU / mL). The cultures were inoculated at 1:100 in 90% serum (90% serum + 10% LB). After 4 h incubation, live bacterial counts were determined by plating the appropriate dilutions on LB agar plates.

[0234] Confocal microscopy. For microscopic analysis, the strains were grown in the medium for the indicated time. Next, the medium was removed, and the cell pellet was washed twice with PBS. Afterward, the pellet was resuspended in 20 μg / mL Hoechst (Thermo Fisher, MA, USA) and incubated at room temperature for 20 min. Subsequently, the pellet was incubated with 20 μg / mL FM4-64 (Biorigin, Beijing, China) at 4℃ for 1 min. A 5 μL aliquot of the culture was then immobilized on adhesion microscope slides. After air-drying, the slides were cover-slipped and imaged using the confocal laser scanning microscopy platform Zeiss LSM880 and LSM800 (Oberkochen, Germany).

[0235] Transmission electron microscopy analysis. First, the strains were cultured in LB or 90% NAS for 2 h at 37℃ with continuous shaking at 180 rpm. Next, the cultures were harvested by centrifugation at 5000 × g for 5 min before being washed twice with double distilled water and fixed in 2.5% glutaraldehyde. Subsequently, the specimens were dehydrated in propylene oxide for 10 min and embedded in an epoxy resin. Lastly, all samples were sent to the Harbin Veterinary Research Institute (Harbin, China) for electron microscopy analysis (Hitachi H-7650, Tokyo, Japan).

[0236] Flow cytometry analysis. The strains (5 × 106CFU) were incubated at 37℃ in 50% NHS for different periods. Next, the cultures were washed thrice with PBS. For the deposition of C3b, bacteria were incubated with FITC-conjugated anti-C3b antibody (Abcam, Cambridge, MA) for 30 min at room temperature. Subsequently, ExPEC XM incubated in HIHS with FITC-conjugated anti-C3b antibody was used as a control. Next, the bacteria were incubated with antibodies against C9 (Abcam, Cambridge, MA, USA) at room temperature for 30 min. Following three washes with PBS, the primary antibody-labeled bacteria were incubated with FITC-conjugated secondary antibodies (Abcam,Agent Ref. P14443WO00 52 Cambridge, USA) for 30 min at room temperature. Next, the bacteria labeled with FITC-conjugated secondary antibodies, without incubation with primary antibodies, were used as controls. Lastly, the surface deposition of molecules was analyzed using a multiparameter flow cytometer (Apogee, Hertfordshire, UK).

[0237] Statistical analysis. Survival was analyzed using the Kaplan–Meier survival method with a log-rank (Mantex-Cox) test, and all other binary comparisons were analyzed using the Student's t-test. P-value < 0.05 was considered statistically significant.

Claims

Agent Ref. P14443WO00 53 CLAIMS What is claimed is:

1. An immunogenic composition comprising: an NhaA protein of an extraintestinal pathogenic Escherichia coli (ExPEC) bacteria; and a pharmaceutically acceptable carrier.

2. The immunogenic composition of claim 1, wherein the ExPEC bacteria comprises avian pathogenic E. coli (APEC) bacteria.

3. The immunogenic composition of claim 1, wherein the pharmaceutically acceptable carrier comprises a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combinations thereof.

4. The immunogenic composition of any one of claims 1-3, wherein the NhaA protein comprises one or more amino acid substitutions or deletions so that said protein is not naturally occurring.

5. The immunogenic composition of claim 4, wherein the one or more amino acid substitutions or deletions of the NhaA protein comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO:

2.

6. The immunogenic composition of claim 5, wherein the one or more amino acid substitutions or deletions of the NhaA protein comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO:

2.

7. An immunogenic composition comprising: a mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene; and a pharmaceutically acceptable carrier.

8. The immunogenic composition of claim 7, wherein the ExPEC bacteria comprises avian pathogenic E. coli (APEC) bacteria.Agent Ref. P14443WO00 54 9. The immunogenic composition of claim 7, wherein the disruption of the endogenous nhaA gene comprises deletion of the nhaA gene.

10. The immunogenic composition of claim 7, wherein the disruption of the endogenous nhaA gene results in complete inactivation of the NhaA protein.

11. The immunogenic composition of any one of claims 7-10, wherein the disruption of the endogenous nhaA gene comprises at least one mutation to loop-2, loop-3, loop-4, loop-5, loop-6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein.

12. The immunogenic composition of any one of claims 7-10, wherein the mutant ExPEC bacteria exhibits attenuated virulence as compared to wild-type ExPEC bacteria.

13. The immunogenic composition of any one of claims 7-10, wherein the disruption of the endogenous nhaA gene comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO:

2.

14. The immunogenic composition of claim 13, wherein the at least one mutation comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO:

2.

15. The immunogenic composition of claim 13, wherein the at least one mutation results in attenuated virulence of the mutant ExPEC bacteria as compared to wild-type ExPEC bacteria.

16. The immunogenic composition of any one of claims 7-10, wherein the mutant ExPEC bacteria is a live, attenuated bacteria.

17. The immunogenic composition of any one of claims 7-10, wherein the pharmaceutically acceptable carrier comprises a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combinations thereof.

18. A method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject, comprising: administering to the subject an immunogenic composition comprising a mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene.Agent Ref. P14443WO00 55 19. The method of claim 18, wherein the ExPEC bacteria comprises avian pathogenic E. coli (APEC) bacteria.

20. The method of claim 18, wherein the disruption of the endogenous nhaA gene comprises deletion of the nhaA gene.

21. The method of claim 18, wherein the disruption of the endogenous nhaA gene results in complete inactivation of the NhaA protein.

22. The method of claim 18, wherein the disruption of the endogenous nhaA gene comprises at least one mutation to loop-2, loop-3, loop-4, loop-5, loop-6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein.

23. The method of claim 18, wherein the mutant ExPEC bacteria exhibits attenuated virulence as compared to wild-type ExPEC bacteria.

24. The method of claim 18, wherein the disruption of the endogenous nhaA gene comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO:

2.

25. The method of claim 24, wherein the at least one mutation comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO:

2.

26. The method of any one of claims 18-25, wherein the at least one mutation results in attenuated virulence of the mutant ExPEC bacteria as compared to wild-type ExPEC bacteria.

27. The method of claim any one of claims 18-25, wherein the mutant ExPEC bacteria is a live, attenuated bacteria.

28. The method of claim any one of claims 18-25, wherein the subject is a poultry animal, mammal, or human.

29. The method of any one of claims 18-25, wherein administration of the mutant ExPEC bacteria immunizes the subject against a pathology caused by ExPEC bacteria.Agent Ref. P14443WO00 56 30. The method of claim any one of claims 18-25, further comprising pharmaceutically acceptable carrier.

31. The method of claim any one of claims 18-25, wherein the pharmaceutically acceptable carrier comprises a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combinations thereof.

32. The method of claim any one of claims 18-25, wherein the immunogenic composition is administered intranasally, intramuscularly, intradermally, subcutaneously, or orally.

33. The method of any one of claims 18-25, wherein the immunogenic composition is administered in a single dose.

34. The method of any one of claims 18-25, wherein the immunogenic composition is administered in two or more doses.

35. A method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject, comprising: administering to the subject an NhaA protein of an extraintestinal pathogenic Escherichia coli (ExPEC) bacteria; and a pharmaceutically acceptable carrier.

36. The method of claim 35, wherein the NhaA protein comprises one or more amino acid substitutions or deletions so that said protein is not naturally occurring.

37. The method of claim 35, wherein administration of the NhaA protein immunizes the subject against a pathology caused by ExPEC bacteria.

38. The method of any one of claims 35-37, wherein the subject is a poultry animal, mammal, or human.

39. The method of any one of claims 35-37, wherein the pharmaceutically acceptable carrier comprises a diluent, adjuvant, antimicrobial agent, preservative, inactivating agent, or combinations thereof.Agent Ref. P14443WO00 57 40. A mutant extraintestinal pathogenic Escherichia coli (ExPEC) bacteria comprising a disruption of the endogenous nhaA gene.

41. The mutant ExPEC bacteria of claim 40, wherein the disruption of the endogenous nhaA gene results in complete inactivation of the NhaA protein.

42. The mutant ExPEC bacteria of claim 40, wherein the disruption of the endogenous nhaA gene comprises deletion of the nhaA gene.

43. The mutant ExPEC bacteria of claim 40, wherein the disruption of the endogenous nhaA gene comprises at least one mutation to loop-2, loop-3, loop-4, loop-5, loop-6, loop-7, loop-8, loop-9, loop-10, and / or loop-11 of the NhaA protein.

44. The mutant ExPEC bacteria of claim 40, wherein the disruption of the endogenous nhaA gene comprises at least one mutation at a position corresponding to position 225, 127, 163, 164, and / or 300 of SEQ ID NO:

2.

45. The mutant ExPEC bacteria of claim 44, wherein the at least one mutation comprises an amino acid substitution H225R, A127V, D163C, D164C, and / or K300C when referenced to SEQ ID NO:

2.

46. The mutant ExPEC bacteria of any one of claims 40-45, wherein the mutant ExPEC bacteria exhibits attenuated virulence as compared to wild-type ExPEC bacteria.

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