CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/045,754, filed September 4, 2014; which is incorporated herein by reference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R21 AI090416 and 2011-67005-30182 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to compositions and methods for treating and preventing sepsis infections. More particularly, the present invention provides compositions and methods for treating and preventing extraintestinal pathogenic Escherichia coli sepsis infections.

[0005] 2. Background

[0006] E. coli strains of significance to humans can be classified according to genetic and clinical criteria into three groups: commensal strains, pathogenic intestinal (enteric or diarrheagenic) strains, and pathogenic extraintestinal strains. Extraintestinal pathogenic Escherichia coli bacteria (ExPEC) exist as commensals in the human intestines but are capable of infecting extraintestinal sites and causing septicemia and other medical conditions. Strains of the ExPEC) group are phylogenetically and epidemiologically distinct from commensal and intestinal pathogenic strains. These are the etiological agents of a diverse spectrum of infections in sites outside of the intestinal tract in humans and animals. Contrary to commensal Escherichia coli (E. coli), ExPEC strains have acquired specific virulence attributes that confer an ability to survive in different niches outside of their normal intestinal habitat in both mammals and birds, of which include colonization, invasion, iron acquisition, serum-complement resistance, antiphagocytic activity, and virulence gene regulation. Critical steps in the process of establishing an ExPEC infection such as meningitis and septicemia include entry into and survival within the bloodstream and internal organs. E. coli sepsis infections often originate from urinary tract infections (UTI), meningitis, proximal gut colonization, wounds and abscesses, and surgical procedures to an infected area or the abdomen.

[0007] E. coli is majorly implicated in bacterial sepsis of both community and nosocomial origins, with a mortality rate ranging from 30-50%, mainly due to the absence of effective antibiotic (ATB) treatments for antimicrobial resistant isolates. Treatment failures due to ATB resistance increase the cost of care and results in prolonged morbidity for patients. As the proportion of elderly and immunocompromised patients is increasing rapidly, the number of E. coli infections will undoubtedly increase, and their treatment will be more challenging. As a result, the prevention of these infections is a pressing concern and prevention strategies are needed to manage these infections in the future. Therefore, there remains a need in the art for improved methods for treating sepsis and other conditions caused by ExPEC bacteria. In addition, there remains a need in the art for improved vaccines that give protective immunity against ExPEC strains.

SUMMARY OF THE INVENTION

[0008] In a first aspect, provided herein is a vaccine composition comprising an antigenically effective amount of at least one recombinant or purified extraintestinal pathogenic Escherichia coli (ExPEC) antigen selected from the group consisting of EcpA, EcpD, IutA, and IroN, and a pharmaceutically acceptable carrier. The recombinant or purified ExPEC antigens can be EcpA and EcpD. The recombinant or purified ExPEC antigens can be IutA and IroN. In some cases, the recombinant or purified ExPEC antigens are EcpA, EcpD, IutA, and IroN. The vaccine composition can further comprise an immunological adjuvant.

[0009] In another aspect, this document provides a unit dosage form comprising a vaccine composition as described herein.

[0010] In a further aspect, provided herein is a method for eliciting an immunological response in a subject against a disease or condition caused by ExPEC bacteria. The method can comprise administering to the subject a therapeutically effective amount of a composition comprising at least one recombinant or purified extraintestinal pathogenic Escherichia coli (ExPEC) antigen selected from the group consisting of EcpA, EcpD, IutA, and IroN. The ExPEC bacteria can be selected from the group consisting of uropathogenic E. coli (UPEC), newborn meningitic E. coli (NMEC), septicaemia associated E. coli (SePEC), and avian pathogenic E. coli (APEC). The composition can be a vaccine composition. The disease or condition can be sepsis. The subject can be a mammal. In some cases, the mammal is human.

[0011] In another aspect, provided herein is a method of protecting a subject from a disease or condition caused by ExPEC bacteria. The method can comprise administering to the subject an antigenically effective amount of a composition comprising at least one recombinant or purified extraintestinal pathogenic Escherichia coli (ExPEC) antigen selected from the group consisting of EcpA, EcpD, IutA, and IroN. The composition can be a vaccine composition. The ExPEC bacteria can be selected from the group consisting of uropathogenic E. coli (UPEC), newborn meningitic E. coli (NMEC),septicaemia associated E. coli (SePEC), and avian pathogenic E. coli (APEC). The disease or condition can be selected from the group consisting of sepsis, bacteremia, septic shock, septicaemia, abdominal sepsis, organ infection; skin infection, blood infection, meningitis, pneumonia, urinary tract infection, and avian colibacillosis. The subject can be a mammal. In some cases, the mammal is human.

[0012] In a further aspect, provided herein is an antibody having specificity for an extraintestinal pathogenic Escherichia coli (ExPEC) antigen selected from the group consisting of EcpA, EcpD, IutA, and IroN. Also provided is a pharmaceutical composition comprising such an antibody.

[0013] These and other features, aspects, and advantages will become better understood upon consideration of the following detailed description, drawings and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a schematic representation of schedules for active immunization and challenge. Mice are vaccinated subcutaneously twice. Humoral responses are monitored using blood serum from mice. Mice are challenged intraperitoneally; both survival and bacterial loads in blood and organs (spleen and liver) are measured. D = Day.

[0015] Figure 2 is (A) a series of western blot images and (B) a table presenting various growth conditions for CFT073. [0016] Figure 3 presents IgG antibody levels induced in BalBc mice immunized to antigens (A) EcpA, (B) EcpD, (C), IutA, (D) IroN, or control (PBS). Data represent IgG antibody levels at day 21 and day 40 post- vaccination for two experiments of similar design. For each experiment, sera were pooled from 3 mice from the same group.

[0017] Figure 4 presents data for total IgG antibody levels induced in BalBc mice immunized with PBS or antigens (A) IutA, (B) IroN, (C) EcpA, and (D) EcpD. Data represent IgG antibody levels as determined individually by IgG-ELISA for each antigen. The values are shown as log IgG total titer. The values are means ± standard deviations for 10 mice from each group. Significant P values compared between groups are represented.

[0018] Figure 5 presents graphs of IgG 1 and IgG2a antibody levels induced in BalBc mice immunized with either PBS or antigens (A, B) IutA,(C, D) IroN, (E, F) EcpA, and (G, H) EcpD, either individually or in combinations. Pooled serum samples of 6 mice immunized with individual antigens or in combinations were analyzed by ELISA for specific antigens.

[0019] Figure 6 demonstrates the effects of vaccination on bacterial loads in organs (spleen, liver) and blood at 48 hours post-challenge. Vaccinated and unvaccinated mice (10/group) were challenged with ~ 3.5 10 ⁷ CFT073 grown O/N in LB + 0.1% glucose + 2,2'bipyridyl shaking. Data are presented as CFU/g or ml (A) or bacterial load level (B); mice were scored based on the level of bacteria in the blood and organs, i.e. No bacteria=0;

hundreds=2; thousands=3; ten-thousands=4; etc. Significant P values compared to PBS group and between all groups (C) are represented.

[0020] Figure 7 presents bacterial clearance data from vaccinated mice and controls. Vaccinated and unvaccinated mice (10 per group) challenged with approximately 3.5xl0 ⁷ CFT073 grown in LB + 2'2' dipyridyl. Blood was drawn 48 hours post-challenge. Red, mice having blood containing bacteria; green, mice having blood cleared from bacteria.

[0021] Figure 8 presents bacterial load data for organs (spleen, liver) and blood at 24 hours post-challenge. Mice were vaccinated with either PBS (control) or with recombinant antigens (rAgs) in different combinations and challenged with approximately 5.5 x 10 ⁷ CFT073 grown in DMEM + Mannose + 2'2' dipyridyl standing for 48 hours CFU/g or ml (24 hours post challenge). P values compared to PBS group are represented.

[0022] Figure 9 depicts the protective capability of rAgs against intraperitoneal challenge with CFT073. (A, B) Data for survival and (C) bacterial loads in liver, spleen, and blood of survived mice in experiment B are presented. Forty-five days after infections, BalB/c mice (n=10/group) were challenged intraperitoneally with CFT073 grown in DMEM + Mannose + dipyridyl standing for 48 hours at either (A) 2.2 x 10 ⁸ or (B) 1.8 xl 0 ⁸ CFU. Mice were regularly monitored for up 48 hours after infections for deaths. Survived mice in (B) were euthanized, necropsied, and the bacterial loads in organs and blood were assessed. Data are representative of two experiments of similar design. P values compared to PBS group (A and B) and between all groups (C) are represented.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Disclosed herein is the discovery of extraintestinal pathogenic E. coli (ExPEC) antigens that, alone or in combination with other antigens, are protective against ExPEC bacterial infections in established lethal and non- lethal murine models of sepsis. As described herein, ExPEC antigens EcpA, EcpD, IutA, and IroN elicited specific humoral responses upon administration. Proteins EcpA and EcpD are the major shaft and polymerized tip adhesin subunits of E. coli common pilus (ECP), respectively. IutA (a ferric aerobactin receptor) and IroN (a salmochelin uptake receptor) are proteins involved in iron acquisition. Without being bound by any theory or mechanism, it is believed that administration of a vaccine of the present invention to a subject stimulates an active immune response against one or more ExPEC antigens.

[0024] Compositions

[0025] Accordingly, provided herein are vaccine compositions useful for treating a subject and immunizing a subject against an ExPEC infection or ExPEC-associated condition such as sepsis. In exemplary embodiments, a vaccine composition comprises an antigenically effective amount of at least one recombinant or purified extraintestinal pathogenic

Escherichia coli (ExPEC) antigen selected from the group consisting of EcpA, EcpD, IutA, and IroN, and a pharmaceutically acceptable carrier.

[0026] As used herein, the term "vaccine" refers to a composition that serves to stimulate an immune response to an ExPEC antigen. As used herein, the terms "therapeutic amount," "effective amount," and "antigenically effective amount" refer to an amount of antigen or vaccine effective to elicit an immune response against an ExPEC antigen present in the composition, thereby treating or preventing ExPEC disease upon administration of the composition to a subject in need thereof. The terms "treat" and "treatment" as used herein refer to either (i) the prevention of infection or re-infection (prophylaxis), or (ii) the reduction or elimination of symptoms of the disease of interest (therapy).

[0027] As used herein, the terms "extraintestinal pathogenic E. coli" and "ExPEC" are used interchangeably herein and refer to pathogenic E. coli strains that invade, colonize, and induce disease in bodily sites outside of the gastrointestinal tract ("extra-intestinal

infections"). ExPEC bacteria include uropathogenic (UPEC) E. coli, newborn meningitic (NMEC) E. coli, septicaemia associated (SePEC) E. coli, and avian pathogenic (APEC) E. coli.

[0028] As used herein, the term "recombinant ExPEC antigen" such as rEcpA, rEcpD, rlutA, and rlroN, refers to the full-length polypeptide sequence, fragments of the reference sequence, or substitutions, deletions and/or additions to the reference sequence, so long as the proteins or fragments thereof retain at least one specific epitope or activity. As used herein, the terms "purified ExPEC antigen" or "isolated ExPEC antigen" such as EcpA, EcpD, IutA, and IroN, are used interchangeably and refer to the full-length polypeptide sequence, fragments of the reference sequence, or substitutions, deletions and/or additions to the reference sequence, so long as the proteins or fragments thereof retain at least one specific epitope or activity. In some cases, the vaccine composition comprises ExPEC antigens EcpA and EcpD. In other cases, the vaccine composition comprises ExPEC antigens IutA and IroN. In yet other cases, the vaccine composition comprises all four antigens: EcpA, EcpD, IutA, and IroN.

[0029] Vaccines provided herein are typically formed by incorporating one or more recombinant or purified ExPEC antigens into pharmaceutically acceptable formulations. The formulations may contain pharmaceutically acceptable adjuvants (such as oils, surfactants, alum), immunostimulatmg agents (such as phospholipids, glycolipids, glycans, glycopeptides, or lipopeptides), and one or more diluents ("excipients"). Examples of diluents suitable for use are water, phosphate buffered saline, 0.15 M sodium chloride solution, dextrose, glycerol, mannitol, sorbitol, dilute ethanol, and mixtures thereof. Pharmaceutically acceptable unit dosage forms of the vaccines can be formulated as solutions, emulsions, dispersions, tablets, or capsules. [0030] In exemplary embodiments, a vaccine composition of the invention further comprises an immunological adjuvant. The terms "immunological adjuvant" and "adjuvant" refer to an agent which acts in a nonspecific manner to increase an immune response to a particular antigen or combination of antigens, thus reducing the quantity of antigen necessary in any given vaccine and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. Such adjuvants and their use are known and available to those who practice in the art and can include, for example, emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such as aluminum hydroxide, chitosan- based adjuvants, and any of the various saponins, oils, and other substances known in the art, such as Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, synthetic

oligonucleotides and combinations thereof (e.g., Schijns et al., Curr. Opi. Immunol. (2000) 12:456 (2000)). Compounds that can serve as emulsifiers herein include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds. Among the synthetic compounds, anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of lauric and oleic acid, the calcium, magnesium and aluminum salts of fatty acids (i.e., metallic soaps), and organic sulfonates such as sodium lauryl sulfate.

[0031] Synthetic cationic agents include, for example, cetyltrimethylammonium bromide, while synthetic nonionic agents are exemplified by glyceryl esters (e.g., glyceryl

monostearate), polyoxyethylene glycol esters and ethers, and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan monopalmitate). Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol. Other suitable adjuvants can be formed with an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil may be a mineral oil, a vegetable oil, or an animal oil.

[0032] The compositions disclosed herein are normally prepared as injectables, either as liquid solutions or suspensions, or as solid forms which are suitable for solution or suspension in liquid vehicles prior to injection. The preparation may also be prepared in solid form, emulsified or the active ingredient encapsulated in liposome vehicles or other particulate carriers used for sustained delivery. For example, the vaccine may be in the form of an oil emulsion, water in oil emulsion, water-in-oil-in-water emulsion, site-specific emulsion, long- residence emulsion, sticky-emulsion, microemulsion, nanoemulsion, liposome, microparticle, microsphere, nanosphere, nanoparticle and various natural or synthetic polymers, such as nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures, that allow for sustained release of the vaccine.

[0033] According to a further aspect there is provided an antibody, or at least an effective binding part thereof, which binds at least one antigen or antigenic polypeptide according to the invention. Preferably, antibodies of the present invention are polyclonal or monoclonal antibodies having specificity to an antigen or polypeptide described herein. Alternatively, the antibody is a chimeric antibody produced by recombinant methods to contain both the variable region of the antibody and an invariant or constant region of a human antibody. In other embodiments, the antibody is humanized by recombinant methods to combine the complimentarity determining regions of the antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.

[0034] In another aspect there is provided a vector which is adapted for the expression of the humanized or chimeric antibodies according to the invention.

[0035] In a yet further aspect, there is provided a cell or cell line which has been transformed or transfected with the vector encoding the humanized or chimeric antibody according to the invention.

[0036] Methods

[0037] According to another aspect, methods for treating an extraintestinal pathogenic E. coli infection are provided. In exemplary embodiments, the present invention provides a method for eliciting an immunological response in a subject against a disease or condition caused by ExPEC bacteria comprises administering to the subject a therapeutically effective amount of a composition comprising at least one recombinant or purified extraintestinal pathogenic Escherichia coli (ExPEC) antigen selected from the group consisting of EcpA, EcpD, IutA, and IroN. The ExPEC bacteria can be uropathogenic E. coli (UPEC), newborn meningitic E. coli (NMEC), septicaemia associated E. coli (SePEC), and avian pathogenic E. coli (APEC). In some cases, the composition administered to the subject is a vaccine composition provided herein. [0038] Diseases and conditions appropriate for treatment according to methods of the invention include, without limitation, sepsis, bacteremia, septic shock, septicaemia, organ infection; skin infection, blood infection, meningitis, pneumonia, cellulitis, and urinary tract infections.

[0039] Appropriate subjects for the methods of treatment include, without limitation, humans diagnosed or suspected of having sepsis or septic shock. It will also be apparent that vaccines or antigenic polypeptides are effective at alleviating conditions in subject other than humans, for example and not by way of limitation, domesticated animals, livestock, and horses.

[0040] Also provided are methods for protecting a subject from developing an

extraintestinal pathogenic E. coli infection. Prophylactic protection is best provided by active immunization, or vaccination, rather than by passive immunization. Accordingly, in exemplary embodiments, a method of protecting a subject comprises administering to the subject a vaccine of the invention. Appropriate subjects for such methods include, without limitation, humans at high risk of developing sepsis or septic shock.

[0041] For human use, the vaccines are preferably administered parenterally, usually via subcutaneous or intramuscular routes of injection. Alternatively, they may be administered intraperitoneally, intravenously, or by inhalation. In general, the vaccine of the present invention is formulated so that a dose of vaccine can be administered in a volume between 0.1 ml and 0.5 ml. The vaccine dosage, the number of doses given to an individual, and the vaccination schedule depend on the antigenicity and immunogenicity of the antigens and on other known pharmaceutical considerations such as the age and body weight of the individual.

[0042] Diseases and conditions against which a subject can be immunized according to a method of the invention include, without limitation, sepsis, bacteremia, septic shock, septicaemia, abdominal sepsis, organ infection; skin infection, blood infection, meningitis, pneumonia, urinary tract infection (pyelonephritis, cystitis, peritonitis), and avian

colibacillosis (airsacculitis, cellulitis, salpingitis, omphalitis, pericarditis, perihepatitis).

[0043] It will also be apparent that vaccines or antigenic polypeptides are effective at preventing conditions in subject other than humans, for example and not by way of limitation, poultry, domesticated animals, livestock, and horses. [0044] In a yet further aspect, the present invention provides a method for the production of a humanized or chimeric antibody according to the invention comprising: (i) providing a cell transformed or transfected with a vector which comprises a nucleic acid molecule encoding the humanized or chimeric antibody according to the invention; (ii) growing said cell in conditions conducive to the manufacture of the antibody; and (iii) purifying the antibody from the cell, or its growth environment. In some cases, methods of the invention involve use of a hybridoma cell line to produce a monoclonal antibody as described herein. A hybridoma cell-line producing monoclonal antibodies can be obtained by: (i) immunizing an immunocompetent mammal with a recombinant or purified ExPEC antigen as described herein; (ii) fusing lymphocytes of the immunized immunocompetent mammal with myeloma cells to form hybridoma cells; (iii) screening monoclonal antibodies produced by the hybridoma cells of step (ii) for binding activity to the antigens of (i); (iv) culturing the hybridoma cells to proliferate and/or to secrete the monoclonal antibody; and (v) recovering the monoclonal antibody from the culture supernatant. Preferably, the immunocompetent mammal is a mouse or rat. The production of monoclonal antibodies using hybridoma cells is well-known in the art. The methods used to produce monoclonal antibodies are disclosed by Kohler and Milstein in Nature 256:495-497 (1975) and also by Donillard and Hoffman, "Basic Facts about Hybridomas" in Compendium of Immunology V.II ed. by Schwartz, 1981, which are incorporated by reference herein as if set forth in their entirety.

[0045] Articles of Manufacture

[0046] Also provided are unit dosage forms comprising a vaccine composition described herein.

[0047] In a further aspect, the use of antibodies for manufacture of a medicament for the treatment of Ex EC-associated septicaemia or another disease or condition disease caused by an extraintestinal pathogenic bacterium is provided.

[0048] Although the embodiments are described in considerable detail with reference to certain methods and materials, one skilled in the art will appreciate that the disclosure herein can be practiced by other than the described embodiments, which have been presented for purposes of illustration and not of limitation. Therefore, the scope of the appended claims should not be limited to the description of the embodiments contained herein. EXAMPLES

[0049] Example 1 : Antigen Combinations Elicit Protective Immune Responses Against E. coli Sepsis

[0050] Four antigen candidates of the E. coli common pilus (EcpA, EcpD) and iron uptake systems (IutA, IroN) were tested by active immunization to determine their ability to elicit specific humoral responses (Thl/Th2) and the protective efficacy of these antigens, singularly and in combinations thereof, against a PBS negative control, in established lethal and non-lethal murine models of sepsis.

[0051] I. Materials and Methods:

[0052] Antigen Preparation and Expression: Genes of the selected antigens (Table 1) were amplified by PCR and cloned into pET-101/D-TOPO® vectors to generate proteins fused with His6 at the C terminus of the protein sequence. Vector DNAs were then introduced into BL21 Star (DE3) expression E. coli (Invitrogen). Following induction of protein expression using IPTG (isopropylthio-P-galactoside), the crude recombinant antigens (rAgs) were purified by affinity chromatography on a Ni- NTA gel matrix under denaturing conditions. Elution fractions were analyzed by SDS-PAGE gel electrophoresis, followed by western blot using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA) using an Anti-His (C-term)-HRP Antibody (Invitrogen). Elution fractions were then concentrated by ultrafugation (Amicon Ultra; Millipore, Billerica, MA), until a protein concentration of greater than 1 mg/ml was achieved. Protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL) and proteins were stored in liquid nitrogen until use.

[0053] Bacterial Challenge Strain: The strain CFT073, isolated from the blood and urine of a hospitalized patient with acute pyelonephritis (Welch et al., PNAS 99: 17020-17024 (2002)) (Table 1), was used as the challenge strain in all experiments described in section below. Unless otherwise stated, bacteria were routinely grown in Luria Bertani (LB) broth or on MacConkey agar supplemented with 0.1% glucose and 1% lactose respectively at 37°C. Strains were stored as stock cultures at -80°C in peptone-glycerol medium.

[0054] Vaccination and Challenge: The vaccination and challenge schedule is shown in Figure 1. Six-week-old female BALB/c mice (Charles River, Wilmington, MA) were subcutaneously injected with purified recombinant protein in Phosphate buffered saline (PBS) (emulsified in Montanide™ ISA 71 VG in a 70:30 w/w ratio) with a total volume of 150 μΐ (Table 2). On day 23, the mice were boosted with lower concentration of antigens in a total volume of 75 μΐ (Table 2). On day 40, mice were bled by the superficial temporal vein technique (cheek bleed), blood sera were extracted and stored at -80°C until processed for serology to assess the humoral antibody responses.

[0055] On day 42, groups of mice were challenged intraperitonially (i.p.) with CFT073 grown in two different conditions (Table 3), in which the bacteria either expressed IutA and IroN but not Ecp, or expressed all four antigens (FIG. 2). In the non-lethal challenge, necropsies were performed After 24 or 48hrs post challenge (FIG. 1), with enumeration of CFU/ml/g recovery in heparinized whole blood, spleens and livers. In the lethal challenge, death was recorded for 48 hours after inoculation; mice that survived to challenge were euthanized and necropsied (FIG. 1). Blood and organs (spleen and liver) were recovered and processed for enumeration of CFU/ml/g.

[0056] Evaluation of Serum Antibody Titers: Serum immunoglobulin endpoint titers were quantified using indirect ELISA protocols. Briefly, ELISAs were performed by coating individual rAgs (2 μg/ml) at 4°C overnight (ON) to Nunc 96 well microtiter plates (Sigma) using pH 9.6 bicarbonate coating buffer. The next day and between all subsequent steps, plates were washed 3 times with 350 μΐ of phosphate buffered saline (PBS) containing 0.1% Tween 20 ("PBST"), using an automated plate washer (BioStack EL406, BioTek, Winooski, VT). Following a one hour block in 2% bovine serum in PBS at room temperature (RT), immune sera or protein standards were two-fold diluted in PBS to the endpoint titer, and incubated for one hour at RT. A goat anti-mouse biotin-coupled secondary antibody (Southern Biotech) was added to the microplates and incubated at RT for one hour before addition of alkaline phosphatase conjugated Streptavidin. After 30 minutes incubation at RT, wells were read using a microtiter plate spectrophotometer (Spectramax M2, Molecular Devices, Sunnyvale CA). Endpoint titers were calculated as the dilution giving an OD ₄₀ 5 _nm two times that for the reagent or unimmunized (PBS only) animal control. The endpoint titers of total IgG, as well as IgGl and IgG2a isotypes were quantified against each antigen respectively in order to determine T helper response involvement.

[0057] Statistical Analysis: Comparison analysis of bacterial clearance and ELISA data was performed using Student's t tests. Survival assay analysis was performed using the Logrank test followed by Student's t tests. P values of <0.05 were considered statistically significant. For all statistical analysis, the software application GraphPad Prism version 6 was used (GraphPad Software, San Diego California USA; available at graphpad.com on the

World Wide Web).

Table 1. Bacterial Strain and Antigens

Strains/rAgs Relevant characteristics Ref.

E. coli

CFT073 06:K2:H1 ST73 (B2), from blood/patient with acute Welch et al, PNAS

pyelonephritis 99: 17020-24 (2002)

Ags

EcpA Major shaft subunit of E. coli common pilus (ECP). Rendon et al, PNAS

104: 10637-42 (2007)

EcpD Polymerized tip adhesin subunit of E. coli common Garnett e? al., PNAS

pilus (ECP). 109:3950-55 (2012)

IutA A ferric aerobactin receptor used for iron acquisition.. Mellata et al, PLoS One

4:e4232 (2009)

IroN Salmochelin uptake receptor used for iron acquisition. Mellata et al, PLoS One

4:e4232 (2009)

Table 2. Antigen Concentration Per Dose

Vaccination Time Antigen combinations and concentration Final

Individual Two Four antigens volume* antigens antigens Low dose high dose

1 ^st vaccination Day 20 μ _§ 20 μg/each 5 μg/each 20 μg/each 150 μΐ

1 (Total 40 μg) (Total 20 μg) (Total 80 μg)

2 ^nd vaccination Day 10 μ _§ 10 μg/each 2.5 μg/each 10 μg/each 75 μΐ

21 (Total 20 μg) (Total 10 μg) (Total 40 μg)

Table 3. Evaluating Vaccine Efficacy

Mouse Bacterial challenge Necropsy# Deaths Results sepsis model Presented

Growth Antigens CFU time post- Organs/fluids Conditions expressed during challenge for bacterial

* challenge loads

Non-lethal I IutA, IroN 3.5 X 48 h Liver, spleen, No Figures 5

10 ⁷ blood and 6

II IutA, IroN, EcpA, 5.5 X 24 h Liver, spleen, No Figure 7

EcpD 10 ⁷ blood

Lethal II IutA, IroN, EcpA, 2.2 X N/A N/A Yes Figure 8A

EcpD 10 ⁸

II IutA, IroN, EcpA, 1.8 X 50 h Liver, spleen, Yes Figures 8B

EcpD 10 ⁸ blood and 8C

* I, LB + 0.1% glucose + 2,2'bipyridyl shaking for O/N at 37°C; II, DMEM + Mannose + 2'2' dipyridyl standing for 48 hours at 28°C, #, done on survivors only; N/A, not applicable

[0058] II. Results

[0059] Selected antigens were expressed and purified as follows. We amplified iutA, iroN, ecpA, and ecpD genes (-ATG) from ExPEC %7122 genome DNA by PCR using high fidelity Platinum® Pfx DNA Polymerase (Life Technology). PCR products for iutA (2199 bp), iroN (2178 bp), ecpA (588 bp), and ecpD (1644 bp) were respectively cloned into pET-101/D- TOPO® cloning vectors. Sequences of the amplified genes were verified by DNA sequencing and compared by BLASTn at NCBI to the published sequences NZ_HE962388 (iutA); (iroN); [ecpA (yagZ)]; [ecpD (yagW)]. The sizes of the purified IutA (74 kDa), IroN (78 kDa), EcpA (21 kDa), and EcpD (45 kDa) proteins were confirmed by SDS-PAGE followed by western blot.

[0060] Levels of antigen-specific (EcpA, EcpD, IutA, and IroN) total IgG in the pooled sera of 6 mice from each group was measured at days 21 and 40 after first vaccination using ELISA. IgG antibodies against the four antigens EcpA, EcpD, IutA and IroN were detected in mice immunized with vaccines containing the specific antigens, either solely or in

combination with other antigens. Depending on the nature of the antigens included in the vaccine, their specific total IgG were elicited differently in vaccinated mice. In fact, anti- EcpA and anti-EcpD IgG were detected in mice sera as early as day 21 post- vaccination and persisted or even increased at day 40 post-vaccination (FIG. 3); while anti-IutA and anti-IroN IgG were only detected at day 40 but not at day 21 post- vaccination (FIG. 3). [0061] The levels of antigen-specific IgG elicited in vaccinated mice depended on the nature of the antigens, their combinations with other antigens, and the concentration of proteins in the vaccine dose injected. In the Ecp A groups, vaccination with Ecp A alone elicited significantly lower IgG compared to those elicited in mice vaccinated with

EcpA+EcpD or all antigens at both low and high doses (FIG. 4). Vaccination with

EcpA+EcpD and all antigens at both high/low doses elicited a similarly high level of anti- EcpA IgG in vaccinated mice (FIG. 4).

[0062] In the EcpD groups, vaccination with EcpD alone elicited significantly lower IgG titers than that produced by the EcpA+EcpD group and all antigens at a high dose group. Mice immunized with all antigens together at high dose, induced the highest level of anti- EcpD IgG compared to all other groups. Immunization with all antigens at a lower dose induced a lower level of anti-EcpD IgG, similar to the one of the EcpD group (FIG. 4).

[0063] In the IutA groups, mice vaccinated with IutA, IutA+IroN, or all antigens at low dose had similar levels of anti-IutA IgG, which was significantly lower than that induced by all antigens at high dose (FIG. 4). A similar trend was seen in the level of anti-IroN IgG in the mice vaccinated with IroN, IutA+IroN, or all antigens at high or low doses (FIG. 4).

[0064] Production of IgG 1 and IgG2a isotypes specific to EcpA, EcpD, IutA, and IroN were evaluated in pooled serum of 6 mice per group immunized with vaccines containing specific antigens. Our data show that all four antigens, either alone or in combination with other antigens, produced both IgGl and IgG2a isotypes in vaccinated mice (FIG. 5). Two antigens, i.e. EcpA and IutA, produced similar levels of IgGl and IgG2a, whereas EcpD and IroN produced different levels of these two IgG isotopes. A higher level of IgGl than IgG2a was induced in mice vaccinated with EcpD, either alone or in combination with other antigens, whereas a higher level of IgG2a than IgGl was induced in mice vaccinated with IroN, either alone or the three other antigens at high dose (FIG. 5).

[0065] Mouse models of non-lethal and lethal sepsis were used to assess the protective effect of vaccine against challenge with CFT073. In the non-lethal sepsis challenge, mice vaccinated with different combinations of antigens and challenged with CFT073 grown in the conditions that expressed IutA and IroN but not Ecp, had lower levels of bacteria compared to the non-vaccinated mice, especially in mice vaccinated with the four antigens at high dose (FIG. 6A). Although no significant differences were found in CFUs, the differences were significant when comparing mice in their bacterial load levels, especially in spleen and blood (FIG. 6B).

[0066] As shown in Figure 7, the percentage of mice with no bacteria in the blood was higher in the groups vaccinated with all antigens (78%) and EcpA (67%). The groups vaccinated with either IutA, EcpA+EcpD, and IutA+IroN had a slightly higher percentage with no bacteria in the blood (40%>) than the PBS group (30%>), whereas groups vaccinated with EcpD and IroN, respectively, had a slightly lower percentage of mice with no bacteria in the blood (10% and 20% respectively) (FIG. 7).

[0067] In the second experiment of non-lethal sepsis, mice vaccinated with all antigens at a high dose and challenged with CFT073 grown in the conditions that expressed both siderophore receptors (IutA, IroN) and adhesins (EcpA, EcpD), had significantly less bacteria in spleen, liver, and blood compared to the non- vaccinated group (FIG. 8).

[0068] In the lethal sepsis challenge, a lethal dose of the CFT073 challenge strain was grown in the condition to express both siderophore receptors (IutA, IroN) and adhesins (EcpA, EcpD). Mice vaccinated with some antigens/combinations of antigens, including EcpA+EcpD, all antigens at high dose, EcpD, and EcpA respectively had better survivability to the bacterial CFT073 challenge (2.2 x 10 ⁸ CFU) than the non- vaccinated group, with results being significant for the group EcpA+EcpD (FIG. 9A).

[0069] In a second similar experiment, but in which the challenge dose was slightly decreased (1.8 x 10 ⁸ CFU). Data show that all vaccinated groups had better protection than the PBS group. The differences were statistically significant in all groups, including EcpD, EcpA+EcpD, and all antigens at high dose, which had 100% protection, EcpA, IutA+IroN, and all antigens at low dose, which had 70%> protection, and finally in the two groups IroN and IutA, which had 50% protection (FIG. 9B).

[0070] Among the mice that survived, those vaccinated with combinations of two or all antigens had significantly lower bacterial load in liver, spleen and blood than mice vaccinated with individual antigens (FIG. 9C). Mice vaccinated with EcpA+EcpD had significantly less bacteria in the liver than mice vaccinated with IutA+IroN (FIG. 9C).

[0071] III. Discussion

[0072] The data presented herein demonstrate our vaccine's protective efficacy against ExPEC. The significant increases in humoral immunity and decreases in CFU recovery in vaccinated mice depict a first step towards the development of a broadly protective vaccine against SEPEC. Although, there are currently no human vaccines available to prevent

SEPEC/ExPEC infections; in these last years, much progress have been made in identifying potential ExPEC antigens and evaluate some of them for protection in mice. Specific

virulence factors, such as adhesins (P and Type 1 fimbriae) (Langermann et al., Science

276:607-11 (1997); Roberts et al., J. Urology 171 : 1682-85 (2004)) and iron uptake, such as IroN, Hma, IreA, lutA (Table 4) were previously tested against UPEC/SEPEC and showed protection in mice, but some of them failed in phase II clinical.

Table 4. Studies on vaccines that included antigens assayed here

Vaccine and composition Challenge Patent Ref.

Mouse model Bacteria/dose

rAgs (Multi- lutA, IhaA, Intraperitoneal CFT073/3X10 ⁶ N/A Wieser et al.,

Infection and epitope) FyuA, Usp CFU Immun.

IroN, IreA, Intraperitoneal CFT073/3X10 ⁶ N/A 78(8):3432-42

(2010). ChuA CFU

rAgs (whole lutA Transurethral CFT073/1X10 ⁸ U.S. Pat. No. Alteri et al,

PLoS protein) CFU 8, 133,496 Pathogens

IroN Transurethral CFT073/1X10 ⁸ 5(9):el000586

(2009). CFU

[0073] We first measured sera antibody responses to the selected antigen tested either alone or in combination with other antigens. Our data show that depending on the nature of antigens, their antibodies are elicited at different periods of time. While specific EcpA and

EcpD IgG were detected after the first vaccination in mice and increased after the second vaccination, both specific lutA and IroN IgG were only detected after the second vaccination.

[0074] Next, levels of antigen-specific IgG antibodies elicited three weeks after the

second vaccination in mice of all groups were compared. It was determined that the nature, the combination, and the dose of antigens affected the level of IgG elicited in the serum of vaccinated mice. Our data show that vaccination with all four antigens at high dose elicited significantly higher level of antigens-specific IgG antibodies (anti-EcpA, anti-EcpD, anti- IutA, and anti-lroN) compared to those elicited by their respective individual antigens. When vaccinated with two combined antigens, only EcpA+EcpD elicited significantly higher level of specific antigens IgG than their respective individual antigens, whereas vaccination with IutA+IroN elicited the same level of antigens-specific IgG antibodies (anti-IutA and anti- IroN) than those elicited by the respective individual antigens. Finally, when all antigens were combined, the high dose elicited significantly higher level of anti-EcpD, anti-IutA, and anti- IroN IgG antibodies than those elicited by the low dose in the specific groups. The level of anti-EcpA IgG elicited was not affected by the dose.

[0075] Recent studies have suggested that both humoral and cell mediated responses are important in a vaccine against ExPEC, as T-cell response is needed for the clearance of the intracellular bacteria (Sivick and Mobley, Infection and Immun. 78:568-585 (2010)). The determination of IgG2a and IgGl isotypes as markers for Thl (cell mediated immunity) and Th2 (antibody mediated immunity) lymphocytes respectively, was investigated in vaccinated mice. Our data show an induction of both humoral- and cell-mediated immunity in vaccinated mice. While EcpA and IutA equally stimulated Thl and Th2 immune responses, EcpD elicited a predominantly Th2-type response and IroN elicited a predominantly Thl -type response.

[0076] We also examined the protective abilities of our vaccines to E. coli sepsis in lethal and non-lethal mouse models. In general, it was determined that the vaccinated mice were better protected than the non- vaccinated mice, as measured by both bacterial loads in the blood and internal organs in non-lethal and lethal challenges. We determined that Ecp antigens, tested for the first time in this study, confer higher protection than siderophore receptor antigens in the lethal sepsis model using a challenge dose of 1.8 x 10 ⁸ CFU.

Moreover, Ecp antigens were still protective when a higher CFU challenge dose of 2.2 x 10 ⁸ CFU was used, while siderophore receptors were not protective in these conditions. Among all vaccine formulas tested, the combination of all antigens at a high dose conferred the best protection.