STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0001] This invention was made with government support under grant number AI024616 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS-REFERENCED TO RELATED PATENT APPLICATIONS

[0002] The present application claims the benefit of priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Application No. 62/022,999, filed on July 10, 2014, and U.S. Provisional Patent Application No. 62/063,743, filed on October 14, 2014, the contents of which are incorporated herein by reference in their entireties

BACKGROUND

[0003] The field of the invention relates to nontypeable Haemophilus influenzae (NTHi). In particular, the field of the invention relates to compositions and methods for vaccinating against infection by NTHi.

[0004] Otitis media is the most common reason children between the ages of 18 months and five years of age seek medical attention. The cost in medical care and economic loss combined is approximately $3.8 billion per year in the United States. (See O'Brien el al. 2009. New vaccines against otitis media: projected benefits and cost-effectiveness. Pediatrics 123: 1452-1463). Approximately, one third of all cases are caused by nontypeable Haemophilus influenzae (NTHi). (See Casey et al. 2013. Acute otitis media otopathogens during 2008 to 2010 in Rochester, New York. The Pediatric infectious disease journal 32:805-809.). NTHi has become increasing resistant to the currently used antibiotics. In addition to otitis media, this organism is a common cause of acute sinusitis in adults as well as acute in respiratory infection in individuals with chronic obstructive lung disease. A vaccine for prevention has become a high priority of the National Institutes of Health. A search for a vaccine has been hampered by the ability of the organism to change surface characteristics thus evading the human immune response.

[0005] We have recently found that there is a conserved carbohydrate epitope, 3-deoxy-d- manno-2-octulosonic acid (KDO), as a terminal sugar on the lipooligosaccharide (LOS) of NTHi and that this epitope is a target for am monoclonal antibody that can kill NTHi. The terminal KDO epitope is present on 40% of NTHi strains obtained from patients with infections. Furthermore, a detoxified, chimeric lipopolysaccharide (LPS) comprising the terminal KDO may be produced in E. coli. (See U.S. Patent No. 6,743,607, the content of which is incorporated herein by reference in its entirety). Because the terminal KDO is a target for a monoclonal antibody that can kill NTHi, carbohydrate immunogens that comprise a terminal KDO may be used in vaccines, which may be administered to prevent NTHi infection. Furthermore, pharmaceutical compositions comprising antibodies against the terminal KDO may be administered to treat infection by NTHi.

SUMMARY

[0006] Disclosed are compositions and methods for vaccination against nontypeable Haemophilus influenzae (NTHi) that utilize 3-deoxy-d-manno-2-octulosonic acid (KDO) as an antigen or immunogen or that utilize chimeric molecules comprising KDO as an antigen or immunogen. The disclosed compositions may be administering in methods for vaccinating a subject in need thereof against infection by NTHi.

[0007] The disclosed methods may include administering an oligosaccharide as an antigen or immunogen to a subject in need thereof, where the oligosaccharide comprises KDO as a terminal sugar residue. In the oligosaccharide that is administered in the disclosed method, the terminal KDO is attached to another sugar, which may include but is not limited to N- acetyllactosamine (LacNAc), galactose (Gal), N-acetylglucosamine (GlcNAc), and glucose (Glc). The terminal KDO may be attached by any suitable oligosaccharide linkage, which may include but is not limited to an α1→ 2 linkage, an α1→ 3 linkage, an α2→ 3 linkage, an α1→ 4 linkage, a β1→ 2 linkage, a β1→ 3 linkage, and a β1→ 4 linkage. In some embodiments, the terminal KDO of the administered oligosaccharide is attached to LacNAc via an αl→ 4 linkage. In other embodiments, the terminal KDO of the administered oligosaccharide is attached to galactose via an α2→ 3 linkage, and optionally the galactose is attached to an N- acetylgalactosamine via a linkage (i.e., where the three terminal residues of the oligosaccharide comprise KDO a2→ 3 galactose β1→ 4 N-acetylglucosamine).

[0008] The disclosed methods may include administering a lipooligosaccharide (LOS) that comprises an oligosaccharide having a terminal KDO. The LOS may be detoxified prior to being administering in the disclosed methods (e.g., by treating the lipid A moiety of the LOS with an alkaline phosphatase to dephosphorylate the lipid A moiety).

[0009] The disclosed methods may include administering a polysaccharide that comprises an oligosaccharide having a terminal KDO. For example, the disclosed methods may include administering a lipopolysaccharide (LPS) comprising a polysaccharide that comprises an oligosaccharide having a terminal KDO. The LPS may be a chimeric LPS and may be detoxified prior to being administering in the disclosed methods.

[0010] In the disclosed methods, the administered oligosaccharide may be conjugated to a peptide, polypeptide, or protein. In some embodiments, the peptide, polypeptide, or protein comprises an antigen or immunogen from NTHi (e.g., an antigen or immunogen selected from the group consisting of outer membrane protein (OMP) of NTHi, surface virulence factor protein D of NTHi, protein E of NTHi, heme utilization protein, protective surface immunogen Dl5, heme binding protein A, or a mixture of these proteins).

[0011] The oligosaccharide administering in the disclosed methods may comprise a mixture of oligosaccharides, some of which mixture of oligosaccharides comprise a terminal KDO, and some of which mixture of oligosaccharides do not comprise a terminal KDO. In some embodiments, preferably the majority of oligosaccharides in the mixture of oligosaccharides comprise a terminal KDO, for example, where at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the oligosaccharides of the mixture comprise KDO as a terminal sugar.

[0012] In the disclosed methods, the oligosaccharide comprising a terminal KDO may be formulated as a vaccine for inducing an immune response against NTHi, the vaccine comprising the oligosaccharide comprising a terminal KDO as an antigen or immunogen and a carrier for delivering the immunogen. The vaccine preferably may be administered to a subject in need thereof in order to induce an antibody response against NTHi.

[0013] The vaccines contemplated herein may comprise additional components for inducing innate or adaptive immunity. In some embodiments, the vaccines may comprise an adjuvant.

[0014] The vaccines contemplated herein may comprise an oligosaccharide comprising a terminal KDO as a first antigen or immunogen and further may comprise a second antigen or immunogen for vaccinating against NTHi. For example, suitable second antigens or immunogens may be selected from an outer membrane protein (OMP) of NTHi, surface virulence factor protein D of NTHi, protein E of NTHi, heme utilization protein, protective surface immunogen D15, heme binding protein A, or a mixture of these proteins.

[0015] Antibodies that bind to a terminal KDO and kill NTHi also are disclosed herein. Because the antibodies have been shown to kill NTHi, the antibodies may be formulated as a pharmaceutical composition comprising the antibodies and a carrier. Typically, the antibodies of the pharmaceutical compositions are monoclonal antibodies which may be humanized monoclonal antibodies. The pharmaceutical compositions comprising the antibodies may be administered to a subject in need thereof in order to treat infection by NTHi.

BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1. Structure of 3-deoxy-d-manno-2-octulosonic acid (KDO).

[0017] Figure 2. One embodiment of one immunogen comprising a terminal KDO residue.

[0018] Figure 3. One embodiment of chimeric immunogen having a terminal structure "R" with a terminal KDO residue.

[0019] Figure 4. One embodiment of chimeric immunogen having a terminal structure "R" with a terminal KDO residue. [0020] Figure 5. The presence of the 6E4 epitope on the surface of NTHi correlates with killing activity.

DETAILED DESCRIPTION

[0021] The disclosed subject matter further may be described utilizing terms as defined below.

[0022] Unless otherwise specified or indicated by context, the terms "a", "an", and "the" mean "one or more." For example, an "antigen" or "immunogen" should be interpreted to mean "one or more antigens" or "one or more immunogens," respectively.

[0023] As used herein, "about", "approximately," "substantially," and "significantly" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" and "approximately" will mean plus or minus <10% of the particular term and "substantially" and "significantly" will mean plus or minus > 10% of the particular term.

[0024] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising" in that these latter terms are "open" transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term "consisting of," while encompassed by the term "comprising," should be interpreted as a "closed" transitional term that limits claims only to the recited elements succeeding this transitional term. The term "consisting essentially of," while encompassed by the term "comprising," should be interpreted as a "partially closed" transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.

[0025] As used herein, the term "subject" may be used interchangeably with the term "patient" or "individual" and may include an "animal" and in particular a "mammal." Mammalian subjects may include humans and other non-human primates, domestic animals, farm animals, and companion animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0026] A "subject in need thereof is intended to include a subject having or at risk for developing an infection by Haemophilus influenzae. In particular, a "subject in need thereof is intended to include a patient having or at risk for developing an infection by nontypeable Haemophilus influenzae (NTHi).

[0027] The compositions and methods disclosed herein include and/or utilize an oligosaccharide comprising a terminal 3-deoxy-d-manno-2-octulosonic acid (KDO) residue. (See Figure I ). The lipooligosaccharide (LOS) of NTHi are known to have a common "deep core" structure which includes a phosphorylated KDO residue conjugated to lipid A. The deep core KDO residue serves to anchor the LOS on the membrane of NTHi and the other sugar residues shield the deep core KDO residue from being exposed at the surface of NTHi. As such, the deep core KDO residue is not exposed as an immunogen in intact bacteria. Therefore, as utilized herein, the phrase "terminal KDO residue" is meant to exclude the deep core KDO residue.

[0028] In addition to the deep core KDO residue, the present inventors have determined that the LOS of NTHi also includes a terminal KDO residue which may function as an immunogen. (See also Spinola el al., "Cloning and Expression in Escherichia coli of a Haemophilus influenzae Type b Lipooligosaccharide Synthesis Gene(s) That Encodes a 2-Keto- 3-Deoxyoctulosonic Acid Epitope," Infection and Immunity, June 1990, p. 1558- 1564; and Kwaik el a/., "Analysis of Haemophilus influenzae type b lipooligosaccharide-synthesis genes that assemble or expose a 2-keto-3-deoxyoctulosonic acid epitope," Molecular Microbiology ( 1991 ) 5(10), 2475-2480; the contents of which are incorporated herein by reference in their entireties). As demonstrated by Spinola el al. and Kwaik el al., the monoclonal antibody 6E4, which reacts with NTHi LOS, recognizes an epitope comprising KDO. (See e.g., Spinola el al., Figure 4, showing that KDO competes for binding to Mab 6E4). [0029] Spinola el al. and Kwaik et al. disclose methods for preparing immunogens suitable for use in the disclosed methods. The disclosed methods include transfecting a bacterial host (e.g., E. coli) with a plasmid comprising genes that are involved in LOS biosynthesis in NTHi (see, e.g., plasmid pGEMLOS-4), including genes encoding enzymes that facilitate the addition of a terminal KDO to the LOS of NTHi such as lipopolysaccharide genes (Isg). In particular, the bacterial hosts may be transfected with plasmids that express LsgB. LsgB has been demonstrated to function as a sialyltransferase. (See Jones el al., "Haemophilus influenzae type b strain A2 has multiple sialyltransferases involved in lipooligosaccharide sialylation," J. Biol. Chem., 2002 Apr 26;277( I7): 14598-61 1 ). However, the present inventors have found that when sialic concentration is low, LsgB uses cytidine 5'-monophospho-3-deoxy-D-manno-2- octulosonic acid (CMP-KDO) as a substrate and adds a terminal KDO residue to the LOS acceptor. The present inventors also have found that the free sialic acid concentration in most body fluids of animals is low enough that KDO is added to the LOS acceptor as a terminal residue by LsgB. Therefore, transfected bacterial hosts that express LsgB may be grown under low sialic acid conditions, and as such, the transfected bacterial hosts will express an LOS or a chimeric LOS/LPS that includes a terminal KDO residue. Methods for producing complex carbohydrates such as chimeric LOS/LPS in host cells are also disclosed in U.S Patent No. 6,743,607, the content of which is incorporated by reference in its entirety. Suitable immunogens which may be prepared by the aforementioned methods of preparation and which may be utilized in the disclosed vaccination methods are illustrated in Figures 2-4.

[0030] The vaccines contemplated herein may comprise an oligosaccharide comprising a terminal KDO as a first immunogen and further may comprise a second immunogen for vaccinating against NTHi. For example, suitable second immunogens may be selected from an outer membrane protein (OMP) of NTHi (e.g., PI , P2, P3, P4, P5, and/or P6), surface virulence factor protein D of NTHi, protein E of NTHi, heme utilization protein, protective surface immunogen DI 5, heme binding protein A, or a mixture of these proteins. (See e.g., Roier et al., "Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice," PLoS One 7(8): e42664. Doi: IO.1371/journal.pone.0042664). For example, a commercial vaccine called (PHiD-CV) (Synflorix™) is a 10-valent pneumococcal non-typeable Haemophilus influenzae protein D- conjugate vaccine that includes ten serotype-specific polysaccharides of Streptococcus pneumoniae, eight of which are conjugated indiv idually to a nonlipidated cell-surface lipoprotein (protein D) of non-typeable H. influenzae and two of which are conjugated to nontoxic tetanus or diphtheria toxoid carrier proteins.

[0031] As used herein, the phrase "treating or preventing infection by NTHi in a subject in need thereof" includes, but is not limited to, preventing or inhibiting infection by NTHi in the subject, preventing or inhibiting an increase in bacterial load in the subject, and/or causing a decrease in the bacterial load in a subject.

[0032] An "effective amount" of an oligosaccharide, antigen, or immunogen is that amount which when administered to a subject induces or elicits an immune response against NTHi in the subject. Immune responses may include antibody-mediated responses, cell- mediated responses, or both. In order to assess the efficacy of the vaccine or immunogenic composition, the immune response may be assessed by measuring antibody induction to particular epitopes of the oligosaccharide (e.g., the KDO epitope) or by measuring the induction of T-cell responses (e.g., CD8 ^' responses) against the particular epitopes of the oligosaccharide. Antibody responses may be measured by assays known in the art such as Western Blots and ELISA. T-cell responses may be measured, for example, by using tetramer staining of fresh or cultured PBMC, ELISPOT assays, or by using functional cytotoxic assays, which are well- known to those of skill in the art. The efficacy of the vaccine or immunogenic composition (e.g., in a subject at risk for developing an infection by NTHi) also may be assessed by observing the non-occurrence of infection in the subject (e.g., the non-occurrence of infection after immunizing the subject and challenging the subject).

[0033] In some embodiments, the disclosed pharmaceutical compositions may be utilized to illicit an immune response de novo. In further embodiments, the pharmaceutical compositions disclosed herein may be utilized to "potentiate" or "enhance" an immune response. As used herein, "potentiating" or "enhancing" an immune response means increasing the magnitude or the breadth of the immune response. For example, the number of cells that recognize a particular epitope may be increased ("magnitude") or the numbers of epitopes that are recognized may be increased ("breadth") (e.g.. 5-fold, or 10-fold relative to a reference composition).

[0034] Formulation and Delivery of the Compositions

[0035] The pharmaceutical compositions disclosed herein include at least one immunogenic component for inducing or eliciting an immune response against NTHi. The disclosed compositions may be utilized as vaccines or immunogenic compositions for treating or preventing infection by NTHi. The terms "vaccine" and "immunogenic composition" are defined herein in a broad sense to refer to any type of biological agent in an administratable form capable of stimulating an immune response in an animal inoculated with the vaccine or immunogenic composition. An immune response may include induction of antibodies or induction of a T-cell response. Herein, the term "protection" when used in reference to an immunogenic composition or vaccine refers to the amelioration (either partial or complete) of any of the signs or symptoms associated with the disease or condition in question.

[0036] The pharmaceutical compositions disclosed herein include at least one immunogenic component for inducing or eliciting an immune response against NTHi. Immunogenic components may include carbohydrates (e.g., oligosaccharides comprising a terminal KDO), polypeptides, or lipid components. The disclosed pharmaceutical compositions may include a panel or plurality of immunogenic components for inducing or eliciting immune responses against infection by NTHi. For example, the compositions may include a panel or plurality of oligosaccharides, polypeptides, or mixtures thereof. As used herein, a "panel" or "plurality" of components means two or more separate and different components (e.g., two or more separate and different oligosaccharides or polypeptides).

[0037] The pharmaceutical compositions disclosed herein may be formulated as vaccines for administration to a subject in need thereof. Such compositions can be formulated or administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration. [0038] The disclosed pharmaceutical compositions may be formulated for delivery in any suitable manner. For example, the compositions may be formulated for at least one of intramuscular delivery, subdermal delivery, subcutaneous delivery, ocular delivery, oral delivery, intravenous delivery, intraperitoneal delivery, intranasal delivery, or pulmonary delivery.

[0039] The pharmaceutical compositions disclosed herein may be delivered via a variety of routes. Typical delivery routes include parenteral administration (e.g., intradermal, intramuscular or subcutaneous delivery). Other potential routes of delivery include ocular administration, oral administration, intranasal administration, pulmonary administration, intravaginal administration, and intrarectal administration. Formulations of the pharmaceutical compositions may include liquid formulations for parenteral, subcutaneous, intradermal, intramuscular, intravenous, or ocular administration (e.g., injectable administration) such as sterile solutions, suspensions, or emulsions. Formulations of the pharmaceutical compositions also may include liquid formulations (e.g., topical formulations or ingestible formulations) for ocular, oral, nasal, anal, and vaginal administration, including solutions, suspensions, syrups or elixirs. The vaccines may be lyophilized prior to delivery and reconstituted prior to administration.

[0040] It is generally advantageous to formulate the present compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suitable as unitary dosages for a patient; each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier, excipient, or diluent. The specification for the dosage unit forms are dictated by and depend on among other factors (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved; (b) the limitations inherent in the art of compounding such active material for the treatment of disease; and (c) the manner of intended administration of the dosage unit form. In some embodiments, a dose of the immunogenic composition or vaccine includes at least about 10 μg (or at least about 20, 40, 60, 80, or 100 μg) of an oligosaccharide comprising a terminal KDO residue. In other embodiments, a dose of the immunogenic composition or vaccine includes about 1-1000 μg (preferably about 10-100 μg) of an oligosaccharide comprising a terminal KDO residue.

[0041] The present immunogenic composition and vaccines may be formulated with a pharmaceutically acceptable carrier, excipient, or diluent. The forms suitable for injectable commonly include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The formulation should desirably be sterile and fluid to the extent that it can be delivered easily with a syringe. The dosage form should be stable under the conditions of manufacture and storage and typically is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier, excipient, or diluent can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. One possible carrier, excipient, or diluent is a physiological salt solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required panicle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal (sodium ethylmercuri- thiosalicylate), deomycin, gentamicin and the like. In many cases it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions, if desired, can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0042] Sterile solutions may be prepared by incorporating a desired amount of the polypeptide in an appropriate solvent, optionally with various amounts of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the various active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and the freeze-drying technique which yield a powder of the active ingredient (i.e., lyophilized form of the active ingredient) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0043] It may also be advantageous to add a stabilizer to the present compositions. Suitable stabilizers include, for example, glycerol/EDTA, carbohydrates (such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as albumin or casein ) and protein degradation products (e.g., partially hydrolyzed gelatin). If desired, the formulation may be buffered by methods known in the art, using reagents such as alkali metal phosphates, e.g., sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate or potassium dihydrogen phosphate. Other solvents, such as ethanol or propylene glycol, can be used to increase solubility of ingredients in the vaccine formulation or the stability of the solution. Further additives which can be used in the present formulation include conventional antioxidants and conventional chelating agents, such as ethylenediamine tetraacetic acid (EDTA).

[0044] The pharmaceutical compositions may be administered prophylactically or therapeutically. For example, in prophylactic administration the pharmaceutical compositions may be administered in an amount sufficient to induce an antibody response or a T-cell response for preventing infection by NTHi. In another example, in therapeutic applications the pharmaceutical compositions may be administered to a patient in an amount sufficient to induce or elicit a therapeutic effect (e.g., an antibody response or T-cell response, which treats the NTHi infection by reducing bacterial load (i.e., as a "therapeutically effective dose")).

[0045] The compositions included in the vaccine regimen of the invention can be coadministered or sequentially administered with other immunological, immunogenic or vaccine or therapeutic compositions. The compositions may be co-administered or sequentially administered with an adjuvant or other therapeutic or prophylactic agent. For example, the disclosed compositions may be administered together with additional agents for preventing and/or treating infection by NTHi. Additional agents for preventing and/or treating infection by NTHi may include, but are not limited to antibiotics such as ceftriaxone, ceftazidime, cefotaxime, ampicillin-sulbactam, fluoroquinolones, and azithromycin. [0046] Adjuvants

[0047] The term "adjuvant" refers to a compound or a mixture that is present in a vaccine and enhances the immune response to an antigen or immunogen present in the vaccine. For example, an adjuvant may enhance the immune response to a oligosaccharide present in a vaccine as contemplated herein. An adjuvant can serve as a tissue depot that slowly releases the antigen or immunogen and also as a lymphoid system activator that non-specifically enhances the immune response. Examples of adjuvants which may be employed include MPL-TDM adjuvant (monophosphoryl Lipid A / synthetic trehalose dicorynomycolate, e.g., available from GSK Biologies). Another suitable adjuvant is the immunostimulatory adjuvant AS021/AS02 (GSK). These immunostimulatory adjuvants are formulated to give a strong T cell response and include QS-21 , a saponin from Quillay saponaria, the TL4 ligand, a monophosphoryl lipid A, together in a lipid or liposomal carrier. Other adjuvants include, but are not limited to, nonionic block co-polymer adjuvants (e.g., CRLI005), aluminum phosphates (e.g., AIPO4), R-848 (a Th 1- like adjuvant), imiquimod, PAM3CYS, poly (l:C), loxoribine, potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum, CpG oligodeoxy nucleotides (ODN), cholera toxin derived antigens (e.g., CTAI-DD), lipopolysaccharide adjuvants, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions in water (e.g., MF59 available from Novartis Vaccines or Montanide ISA 720), keyhole limpet hemocyanins, and dinitrophenol. Further, the oligosaccharides disclosed herein may be conjugated to a peptide, polypeptide, or protein (e.g., as a carrier) in order to increase immunogenicity of the oligosaccharide. In some embodiments, the oligosaccharides disclosed herein may be conjugated to another immunogen of NTHi which optionally is selected from an outer membrane protein (OMP) of NTHi (e.g., P1, P2, P3, P4, P5, and/or P6), surface virulence factor protein D of NTHi, protein E of NTHi, heme utilization protein, protective surface immunogen D15, heme binding protein A, or a mixture ofthese proteins.

[0048] Prime-Boost Vaccination Regimen [0049] The disclosed composition may be administered as vaccines utilizing a selected "prime-boost vaccination regimen." As used herein, a "prime-boost vaccination regimen" refers to a regimen in which a subject is administered a first composition one or more times (e.g., one time or two or three times with about 2, 3, or 4 weeks between administrations) and then after a determined period of time after having administered the first composition (e.g., about 2 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or longer), the subject is administered a second composition. The second composition may also be administered more than once, with at least 2, 3, or 4 weeks between administrations. The first and second compositions may be the same or different.

[0050] Methods and Kits

[0051] The disclosed compositions may be utilized in methods for immunizing a patient against infection by NTHi. Kits are also contemplated herein including kits for administering the disclosed compositions and kits for making the disclosed compositions. The kits may include one or more components for performing the administration methods disclosed herein. For example, the kits may include one or more of the vaccine or pharmaceutical compositions disclosed herein or components for making or administering the vaccine or pharmaceutical compositions disclosed herein. The vaccine or pharmaceutical compositions or components may be provided in any suitable form (e.g., liquid form or lyophilized form). Kits further may include solvents for resuspending or dissolving a lyophilized oligosaccharide.

EXAMPLE

[0052] The following example is illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0053] A Vaccine for Nontvpeable H. Influenzae

[0054] Abstract [0055] The inventors have found that there is a conserved carbohydrate epitope, 3-deoxy- d-manno-2-octulosonic acid (KDO), as a terminal sugar on the lipooligosaccharide (LOS) of NTHi and that this epitope is a target for antibody that can kill NTHi. This epitope is present on 40% of NTHi strains obtained from patients with infections. The inventors have prepared a detoxified chimeric LPS molecule comprising KDO as a terminal sugar. The inventors propose using this detoxified chimeric LPS molecule as an immunogen to produce antibodies directed against the KDO epitope. The inventors propose that this immunogen would be a suitable component of a vaccine to prevent NTHi infection by strains comprising the KDO epitope.

[0056] Introduction

[0057] Nontypeable Haemophilus influenzae (NTHi) is a major cause of upper respiratory infections. It appears to be the major cause of otitis media (OM), a middle ear infection common among children, as well as sinusitis and bronchial infections. OM is the most common illness for which children visit a physician, receive antibiotics, or undergo surgery (tympanostomy tubes) in the US. OM may result after NTHi enters the eustachian tube of the ear via the throat. The eustathian tube then closes and pus and fluid forms which puts pressure on the eardrum causing pain. The cost of OM treatment alone is estimated to be $5 billion per year in the US.

[0058] Physicians are aware that antibiotic therapy is ineffective in many cases of NTHi infections due to growing resistance. This discovery prompted the use of alternative therapies for OM, such as surgery for patients with repetitive cases and "no treatment" recommendations when possible.

[0059] Vaccines against H. influenzae have been proposed. (See U.S. Patent Nos. 6,743,607; 7,364,739; and European Patent No. 941738. These vaccines generally are drawn to heterogenous lipooligosaccharides (LOS) moieties on the surface of the bacteria. Expression of these heterogeneous oligosaccharide epitopes is important for host— pathogen interactions and therefore is an essential target for regulating the virulence potential of this organism. [0060] The LPS of many bacteria include an outer O-antigen polysaccharide which may be utilized to serotype the bacteria. However, NTHi does not produce a LPS with an O-antigen polysaccharide but rather produces a surface glycolipid with oligosaccharide structures, hence the term, lipooligosaccharide (LOS) rather than lipopolysaccharide (LPS) is used to refer to the oligosaccharide structures on the cell surface of NTHi.

[0061] Common sugar constituents found in LOSs of H. influenzae serotype B & NTHi include Glucose (Glc), galactose (Gal), l-glycero-d-manno-heptose (l,d-Hep), and a deep core 3- deoxy-d-manno-2-octulosonic acid (KDO). KDO is an ulosonic acid of a 2-ketooctose (i.e., an eight carbon sugar), where the "D-manno" prefix indicates that four chiral centers have the same configuration as D-mannose. (See Figure I ). KDO is produced only in bacteria. In addition to Glc, Gal, I ,d-Hep, and KDO, some LOSs also contain N-acetyl-glucosamine (GlcNAc).

[0062] Common non-glycosidic constituents of LOSs include phosphate, phosphoethanolamine (PEA), and acetyl groups. For example, the deep core KDO residue present in H. influenzae LOSs has been found to be phosphorylated in both the serotype b strain and in NTHi strains,

[0063] The H. influenzae LOSs can generally be described as including an anchor of diacyl lipid A attached to a single phosphorylated KDO, an inner core of sugar constituents (i.e., inner core), and an outer core of sugar constituents (i.e., outer core). Structural studies have shown that α-l,d-Hep-( l→2)-α-l,d-Hep-( l→3)-α-l,d-Hep-( l→5)-Kdop (i.e., Heplll-Hepll-Hepl- Kdo) is a common LOS inner core element for H. influenzae strains. Structural variation of these LOSs is due to the various OSs attached to Hepl, HepII, and/or Heplll, to the phosphate and phosphoethanolamine (PEA) substituents on HepII and Heplll, and to a phosphocholine- glucosyl moiety attached to Heplll.

[0064] The common occurrence of infection and high variability found in NTHi makes it a problem for future antibiotic treatment. Therefore, infection prophylaxis is important, particularly in the pediatric population. As a candidate antigen for vaccinating against NTHi infection, the present inventors propose utilizing a unique structure found on the terminus of Haemophilus influenzae LOS, which is an epitope comprising a terminal KDO residue.

[0065] Spinola et al. found that a KDO epitope may be recognized by antibodies against the LOS. (See Spinola et al., "Cloning and Expression in Escherichia coli of a Haemophilus influenzae Type b Lipooligosaccharide Synthesis Gene(s) That Encodes a 2-Keto-3- Deoxyoctulosonic Acid Epitope," Infection and Immunity, June 1990, p. 1558-1564.) Spinola et al. prepared a monoclonal antibody (mAb) against the LOS of NHTi called "6E4." The monoclonal antibody 6E4 was produced by immunizing Balb/C mice with 0.1 ml peritoneally of I0>7 cfu heat killed (65C for l hour) nontypeable H. influenzae in phosphate buffered saline (PBS) on day one followed by immunizations with I0>4 cfu heat killed organisms on day 21 and day 30. The animals were euthanized on day 35 and their spleen removed for recovery of B- cells. The standard fusion procedure for production of hybridomas was then carried out to produce the hybridomas. Initial screening for monoclonals was performed using heat killed bacteria as the antigen in ELISA studies. Fluid from wells which had reactivity to the whole cells was tested in Western blot assay and the nature of the specificity of the antibody determined. Antibodies which reacted to the lipooligosaccharide (LOS) were tested with purified LOS for confirmation. Spinola el al. demonstrated that the MAb 6E4 binds specifically to an epitope of NTHi LOS that comprises a terminal KDO in competition ELISA experiments using free KDO. (See Spinola el al., Figure 4).

[0066] Monoclonal Antibody 6E4 is Bactericidal

[0067] The present inventors tested whether the monoclonal antibody 6E4 was bactericidal in a complement-based method. Thirty-four bacterial strains were grown overnight on sRPMI agar, then scraped from a sparsely populated region of the plate and diluted in Hank's Balanced Salt Solution (HBSS) to a concentration of 10 ⁷ bacteria/ml. Bactericidal assays, modified from a method reported by Zaleski et al. were carried out in 96-well plates with a 50-ul total volume. (See Zaleski et al., "Lipooligosaccharide P(k) (Calalphal -4Galbetal -4Glc) epitope of moraxella catarrhalis is a factor in resistance to bactericidal activity mediated by normal human serum," Infect. Immun. 2000 Sep ;68(9): 5261, the content of which is incorporated herein by reference in its entirety).

[0068] A gamma human serum (AGS) was used as a source of complement without antibody at a 1 : 10 dilution. Monoclonal antibody 6E4 was added at final concentration of I μg/ml Controls containing AGS heat-inactivated for 30 minutes at 56C were included in each experiment. Five microliters of the resuspended bacteria was diluted into 45 microliters of HBSS, and serial 1 : 10 dilutions were made in HBSS. Five microliters of each dilution was pipetted onto sBHl agar and grown overnight at 37°C in 5% CO ₂ . The colonies in these reaction mixtures were counted and used as the initial CFU. Five microliters of the bacterial stock was incubated with AGS and mAb 6E4 for 30 minutes with shaking at 200 rpm in a 37°C incubator. Serial 1 : 10 dilutions of the reaction mixtures were made in HBSS and 5 μl of each dilution was pipetted onto sBHl agar. These were grown overnight at 37°C in 5% CO: and colonies were counted the next day. Killing was assessed by comparing the number of CFU after 30 minutes incubation with AGS and mAb 6E4 with the initial CFU. (See Figure 5). The results illustrate that greater than 50% of the organisms were killed based on binding of the 6E4 mAB to the KDO epitope (black bars). ELISA studies demonstrated that the level of binding of the 6E4 mAB to the bacterial outer membrane generally correlated with the level of killing (grey bars).

[0069] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0027] Citations to a number of references may be made herein. Any cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.