WO2009033269A1

WO2009033269A1 - Novel polysaccharide immunogens from alloiococcus otitidis and synthesis of a glycoconjugate vaccine thereof

Info

Publication number: WO2009033269A1
Application number: PCT/CA2008/001594
Authority: WO
Inventors: Mario Artur Monteiro; Sarif Arar; Atsushi Harimaya
Original assignee: University Of Guelph
Priority date: 2007-09-11
Filing date: 2008-09-11
Publication date: 2009-03-19

Abstract

The application relates to Alloiococcus otitidis cell surface polysaccharides, compositions comprising Alloiococcus otitidis cell surface polysaccharides, synthesis of a glycoconjugate vaccine and includes kits, methods and uses thereof.

Description

Title: Novel Polysaccharide lmmunogens from Alloiococcus otitidis and Synthesis of a Glycoconjugate Vaccine Thereof

[0001] The present application relates to novel cell surface polysaccharides and methods and uses thereof.

BACKGROUND OF THE APPLICATION

[0002] Middle ear infections (otitis media) represent one of the most serious ailments that afflict children (1). The pathogens, Streptococcus pneumoniae, nontypeable Haemophilus influenzae and Moraxella catarrhalis have become well recognized as those being associated with otitis media (2). However, in the late 1980s, Faden and Dryja reported the identification of a new Gram-positive bacterium that showed a strong association with childhood otitis media (3). Subsequently, this unknown cocci was given the name Alloiococcus otitidis (4,5). A limited number of investigations have now furnished data demonstrating a direct connection between A. otitidis infections and otitis media, and in fact A. otitidis is now being more frequently detected in otitis media patients (6-8). Moreover, it has been noted that the incidence of A. otitidis is significantly high in the nasopharynx of children prone to ear infections (9), and that, even upon treatment with antibiotics, in most cases the pervasiveness of A. otitidis remained remarkably high (10). A recent study by Harimaya and co-workers (11) showed evidence of local antibody response against A. otitidis in the middle ear effusions of patients. The observed presence of IgG₁ secretory IgA, lgG2, and IgM against A. otitidis suggested that specific local immune response against A. otitidis is induced during middle ear infection with this organism (11). The complete genome sequence of A. otitidis strain 1104-92 is available in a patent application (12), with defined polynucleotide sequences encoding putative antigenic polypeptides encoded by A. otitidis strain 1104-92 open reading frames being described. SUMMARY OF THE APPLICATION

[0003] The present application discloses novel Alloiococcus otitidis cell surface polysaccharides as well as their covalent chemical structures. These novel polysaccharides are used in immunogenic compositions, and in anti-A otitidis vaccine preparations.

[0004] Accordingly, the present application includes isolated immunogenic Alloiococcus otitidis cell surface polysaccharides.

[0005] In one embodiment, the cell surface polysaccharides comprise

N-acetyl-D-glucosamine (GIcNAc), N-acetyl-D-galactosamine (GaINAc), D- glucuronic acid (GIcA) and optionally, L-glutamic acid (GIu) within their covalent structure.

[0006] In another embodiment, the cell surface polysaccharides comprise repeating trisaccharide units. In another embodiment, the repeating trisaccharide units comprise GIcNAc, GaINAc, GIcA and optionally, GIu. [0007] In one embodiment, the cell surface polysaccharide comprises repeating trisaccharide units of the formula I and II:

→6-GalNAc-1→4-GlcA-1-→3-GlcNAc-1→ (I)

→6-GalNAc-1→4-GlcA-1→3-GlcNAc-1→ (II) 6

4

GIu

[0008] In one embodiment, the cell surface polysaccharide is a compound of the formula (PS-1): [→6)-β-GalNAc-(1 -»4)-[Glu→6]-β-GlcA-(1 →3)-β-GlcNAc-(1 ]„ wherein n is an integer from 1-1000, wherein the 6 carboxyl group of GIc A is optionally substituted with GIu; and immunogenic fragments thereof. [0009] In another embodiment, the cell surface polysaccharides are compounds of the formula PS-2, which comprises phosphate, terminal and 2- substituted glucose, and trisubstitued glycerol within their covalent structure.

[0010] Another aspect of the present application is a cell surface polysaccharide mixture comprising one or more Alloiococcus otitidis cell surface polysaccharides.

[0011] A further aspect of the present application includes conjugation of one or more Alloiococcus otitidis cell surface polysaccharides disclosed herein to one or more carrier molecules. In one aspect, the carrier molecule is a protein. In a further aspect, the carrier molecule is BSA, CRM₁97, MIEP, Dipthteria toxoid, Tetanus toxoid or proteins derived from Bordetella

[0012] Another aspect of the present application is an immunogenic composition comprising one or more Alloiococcus otitidis cell surface polysaccharides. [0013] A further aspect of the present application is a vaccine composition comprising one or more Alloiococcus otitidis cell surface polysaccharides.

[0014] Another aspect of the present application is a kit comprising the cell surface polysaccharides disclosed herein or the cell surface polysaccharide mixture disclosed herein or the immunogenic compositions disclosed herein or vaccine compositions disclosed herein and instructions for use.

[0015] Another aspect of the present application is a method of inducing an immune response against Alloiococcus otitidis in a subject by administering to a subject in need thereof an effective amount of one or more of the cell surface polysaccharides disclosed herein or the immunogenic compositions disclosed herein or the vaccine compositions disclosed herein.

[0016] A further aspect of the present application is a method of treating or preventing Alloiococcus otitidis infection in a subject by administering to a subject in need thereof an effective amount of one or more of the cell surface polysaccharides disclosed herein or the immunogenic compositions disclosed herein or the vaccine compositions disclosed herein.

[0017] An additional aspect of the present application is a method of treating or preventing otitis media in a subject by administering to a subject in need thereof an effective amount of one or more of the cell surface polysaccharides disclosed herein or the immunogenic compositions disclosed herein or the vaccine compositions disclosed herein.

[0018] The present application also includes uses of one or more of the cell surface polysaccharides disclosed herein or the immunogenic compositions disclosed herein or the vaccine compositions disclosed herein to induce an immune response in a subject against Alloiococcus otitidis, to treat or prevent Alloiococcus otitidis infection in a subject, and to treat or prevent otitis media in a subject.

[0019] A further aspect of the present application includes uses of one or more of the cell surface polysaccharides disclosed herein or the immunogenic compositions disclosed herein or the vaccine compositions disclosed herein for the manufacture of a medicament to induce an immune response in a subject against Alloiococcus otitidis, to treat or prevent

Alloiococcus otitidis infection in a subject, and to treat or prevent otitis media in a subject.

[0020] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention will now be described in relation to the drawings in which: [0022] Figure 1 is a spectrum of an ESI/TOF-MS/MS experiment carried out on m/z 1427.08 showing daughter m/z ions of defined composition that revealed the presence of a chain blocks composed of Λ/-acetyl- hexosamine (GaINAc and GIcNAc) and hexuronic acid (GIcA), which could be substituted by a glutamic acid (GIu) residue. The sequences shown contain the GaINAc, GIcNAc, GIcA and GIu designations (the units previously characterized by GLC-MS).

[0023] Figure 2 is an EI-MS spectrum of the methylated cell surface PS showing primary glycosyl oxonium ions and respective secondary m/z ions emanating from the non-reducing-end, which specify the triglycosyl sequence of the cell surface PS. The two trisaccharide structures shown represent the termini blocks present at the non-reducing-end of the cell surface PS.

[0024] Figure 3 is a MALDI-TOF-MS spectrum of the cell surface PS obtained from mild-acid treatment of cells. [0025] Figure 4 is a ¹H NMR spectrum of cell surface PS showing the presence of β anomeric resonances (OH 4.35-4.65), of singlets belonging to methyl groups of Λ/-acetyl-hexosamines (6H 2.06), and of methylene moieties of GIu amino acid (δ_H 1.96) and (δ_H 2.26).

[0026] Figure 5 is a 2D ¹H-¹H COSY spectrum of cell surface PS showing five distinct β anomeric resonances (labeled A, B, C, D and E), and also the spin-system belonging to the GIu residue (W, X, Y and Z).

[0027] Figure 6(A) shows the assignment of carbon resonances of cell surface PS by ¹H-¹³C HSQC (green and blue), and the detection of through- bond glycosyl linkages by ¹H-¹³C HMBC (red). (B) Expansion of the ¹H-¹³C HMBC spectrum displaying region containing the connectivities between the carbonyls of GIu and GIcA and neighbouring protons.

[0028] Figure 7 is a ¹H-¹H NOESY spectrum of cell surface PS showing through-space nOe connectivities that substantiated the glycosyl linkages previously described. [0029] Figure 8 is a MALDI-TOF-MS spectrum of the cell surface PS-

BSA conjugate indicating that, on average, 3 cell surface PSs were covalently appended to a BSA unit in this conjugate. This spectrum also showed the presence of conjugates containing BSA units carrying up to 7 cell surface PSs.

[0030] Figure 9(A) shows the dot blot analysis (measuring human IgG antibody) of A otitidis cell surface polysaccharide with middle ear effusion of otitis media human patients due to A. otitidis infection. Figure 9(B) shows the Western blot analysis (detecting human IgG antibody) of A. otitidis cell surface polysaccharide with middle ear effusion of otitis media human patients due to A. otitidis infection.

[0031] Figure 10 is a GC-MS profile of the sugar composition of an A. otitidis preparation to which IgG, secretory IgA₁ lgG2, and IgM human antibodies from otitis media patients were detected. DETAILED DESCRIPTION OF THE APPLICATION I. DEFINITIONS

[0032] The term "Alloiococcus otitidis" as used herein includes all strains of Alloiococcus otitidis, including for example, ATCC 51267, SS1337, 1514-89, 1515-89, 1518-89, 1996-94, and 2621-96. [0033] The term "isolated" as used herein refers to Alloiococcus otitidis cell surface polysaccharides substantially free of bacterial cell material or extraction solvent when produced from growing bacterial strains of Alloiococcus otitidis.

[0034] As used herein the term "Alloiococcus otitidis cell surface polysaccharides" includes those isolated from bacterial strains of Alloiococcus otitidis, and also includes polysaccharides produced synthetically to have the same structure and/or composition of the Alloiococcus otitidis cell surface polysaccharides disclosed herein. "Produced synthetically" includes for example, cell surface polysaccharides produced from techniques such as recombinant DNA technology, genetic knockout mice and/or chemical synthesis.

[0035] The term "covalent chemical structure" as used herein means the chemical formula for a compound where all groups are linked via covalent bonds.

[0036] As used herein the term "fragment thereof means any portion of the cell surface polysaccharides disclosed herein that retains immunogenic activity against Alloiococcυs otitidis. The fragment may contain one or more if the monosaccharides (sugars) or sugar phosphates that are within the covalent chemical structures of the polysaccharides. Whether or not the fragment retains immunogenic activity may be determined using techniques known in the art.

[0037] As used herein the term "immunogenic" means the ability to elicit an immune response. [0038] As used herein the term "vaccine" refers to a composition that prevents Alloiococcus otitidis infection, treats Alloiococcus otitidis infection and/or reduces shedding of Alloiococcus otitidis.

[0039] The term "therapeutically effective amount", "effective amount" or "sufficient amount" means a quantity sufficient to, when administered to the subject, including a mammal, for example a human, achieve a desired result, for example an amount effect to elicit an immune response in a subject. Effective amounts of therapeutic may vary according to factors such as the disease state, age, sex, weight of the animal. Dosage or treatment regime may be adjusted to provide the optimum therapeutic response. [0040] Moreover, a "treatment" regime of a subject with a therapeutically effective amount may consist of a single administration, or alternatively comprise a series of applications. The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the patient, the concentration and the activity of the polysaccharides, or a combination thereof. It will also be appreciated that the effective dosage of the compound used for the treatment or prevention may increase or decrease over the course of a particular treatment or prevention regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. The compounds of the present disclosure may be administered before, during or after exposure to the bacteria.

[0041] The expression "biologically compatible form in vivo" as used herein means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.

[0042] The term "eliciting an immune response" or "inducing an immune response" as used herein means initiating, triggering, causing, enhancing, improving or augmenting any response of the immune system, for example, of either a humoral or cell-mediate nature. The initiation or enhancement of an immune response can be assessed using assays known to those skilled in the art including, but not limited to, antibody assays (for example ELISA assays), antigen specific cytotoxicity assays and the production of cytokines (for example ELISPOT assays).

[0043] The term "subject" as used herein refers to any member of the animal kingdom, preferably a mammal. In one embodiment, the mammal is a dog, a cat, a hamster, a mouse, a rat, a pig, a horse, cattle or a human being. In another embodiment, the mammal is a pig, a horse, cattle or a human being.

[0044] As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. [0045] "Palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder. [0046] For example, the phrase "treating or preventing Alloiococcus otitidis infection" refers to inhibiting Alloiococcus otitidis infection, preventing Alloiococcus otitidis infection, reducing shedding of Alloiococcus otitidis, decreasing the severity of Alloiococcus otitidis infection or improving signs and symptoms related to Alloiococcus otitidis infection and the phrase "treating or preventing otitis media" refers to inhibiting otitis media, preventing otitis media, decreasing the severity of otitis media or improving signs and symptoms related to having otitis media. The present application also include the treatment or prevention of any disease that is associated with an Alloiococcus otitidis infection. [0047] In understanding the scope of the present application, the term

"comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

II. COMPOUNDS AND COMPOSITIONS OF THE APPLICATION

[0048] As mentioned above, the present application describes the isolation and identification of the chemical structure of Alloiococcus otitidis cell surface polysaccharides. These novel polysaccharides are exposed on the cell surface of Alloiococcus otitidis are used in immunogenic compositions and in carbohydrate-based anti-A otitidis vaccine preparations.

[0049] Accordingly, the present application includes isolated immunogenic Alloiococcus otitidis cell surface polysaccharides. The present application also includes an Alloiococcus otitidis cell surface polysaccharide mixture comprising one or more Alloiococcus otitidis cell surface polysaccharides.

[0050] The present application also includes an immunogenic composition comprising one or more Alloiococcus otitidis cell surface polysaccharides.

[0051] The present application further includes a vaccine composition comprising one or more Alloiococcus otitidis cell surface polysaccharides.

[0052] The present application describes the isolation and characterization of two distinct cell surface Alloiococcus otitidis cell surface polysaccharides. These are designated PS-1 and PS-2 in the present application.

[0053] Using chemical analysis and analytical techniques, such as

NMR and mass spectrometry, it has been determined that the cell surface polysaccharide PS-1 of Alloiococcus otitidis (strain ATCC 51267) comprises repeating trisaccharide units composed of 3-substituted Λ/-acetyl-D- glucosamine (GIcNAc), 6-substituted Λ/-acetyl-D-galactosamine (GaINAc), and 4-substituted D-glucuronic acid (GIcA), of which the majority was amidically decorated with L-glutamic acid (GIu). The cell surface polysaccharide designated as PS-2 was determined to comprise phosphate, terminal and 2-substituted glucose, and trisubstituted glycerol.

[0054] In an embodiment, the cell surface polysaccharides are obtained by isolation from strains of Alloiococcus otitidis bacteria, for example by growing Alloiococcus otitidis bacteria in suitable medium, separating bacterial cells from the medium, extracting cell surface polysaccharides by acid treatment, and purifying the extracted cell surface polysaccharides. [0055] Accordingly, in one embodiment of the present application, the cell surface polysaccharides are isolated from A. otitidis cells by treatment of the cells with mild acid under conditions to cleave cell surface polysaccharides from the cells. In another embodiment, the mild acid is 0.1% to 5% acetic acid, suitable 2% acetic acid. In another embodiment, the cell surface polysaccharides are further purified by dialysis, lyophilization and/or size exclusion chromatography.

[0056] In one embodiment, the cell surface polysaccharides comprise

N-acetyl-D-glucosamine (GIcNAc), N-acetyl-D-galactosamine (GaINAc), D- glucuronic acid (GIcA) and optionally, L-glutamic acid (GIu) within their covalent structure. In another embodiment, the cell surface polysaccharides further comprise one or more glycerol, glucuronic acid, mannose, glucose and galactose within their covalent structure.

[0057] In another embodiment, the cell surface polysaccharides comprise repeating trisaccharide units. In another embodiment, the repeating trisaccharide units comprise GIcNAc, GaINAc, GIcA and optionally, GIu. In another embodiment, the GIcA is non-stoichiometrically substituted with the

GIu. In a further embodiment, the GIu is attached to the GIcA through an amide linkage between the 6-carboxyl group of GIcA and the amine of GIu. In another embodiment, the cell surface polysaccharides further comprise one or more glycerol, glucuronic acid, mannose, glucose and galactose within their covalent structure.

[0058] In one embodiment, the cell surface polysaccharides comprise repeating trisaccharide units of the formula I and II: →6-GalNAc-1→4-GlcA-1→3-GlcNAc-1→ (I)

→6-GalNAc-1→4-GlcA-1→3-GlcNAc-1→ (II)

6

I GIu [0059] In one embodiment, the cell surface polysaccharides are compounds of the formula (PS-1):

[→6)-β-GalNAc-(1→4)-[Glu→6]-β-GlcA-(1→3)-β-GlcNAc-(1]_n wherein n is an integer from 1 to 1000, wherein the 6 carboxyl group of GIc A is optionally substituted with GIu; and immunogenic fragments thereof.

[0060] In one embodiment, n in PS-1 is an integer from 1 to 100, 2 to

100, 10 to 100, 25 to 100, or 5 to 20.

[0061] In another embodiment, the cell surface polysaccharides comprising the trisaccharide units in formula I or II, further comprise one or more glycerol, glucuronic acid, mannose, glucose and galactose.

[0062] In another embodiment, the cell surface polysaccharides of the formula PS-1 further comprise one or more glycerol, glucuronic acid, mannose, glucose and galactose.

[0063] In another embodiment, the cell surface polysaccharides are compounds of the formula PS-2, which comprises phosphate, terminal and 2- substituted glucose, and trisubstituetd glycerol within their covalent structure.

[0064] Another aspect of the present application is a cell surface polysaccharide mixture comprising one or more Alloiococcus otitidis cell surface polysaccharides. In one embodiment, the cell surface polysaccharide mixture comprises: a) cell surface polysaccharide PS-1 disclosed herein; and b) cell surface polysaccharide PS-2 disclosed herein.

[0065] In another embodiment, the present application discloses an immunogenic composition comprising one or more Alloiococcus otitidis cell surface polysaccharides and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or mixtures thereof.

[0066] In an embodiment, the immunogenic composition comprises cell surface polysaccharides comprising N-acetyl-D-giucosamine (GIcNAc), N- acetyl-D-galactosamine (GaINAc), D-glucoronic acid (GIcA) and optionally, L- glutamic acid (GIu) within their covalent structure. In another embodiment, the immunogenic composition comprises cell surface polysaccharides further comprising one or more glycerol, glucuronic acid, mannose, glucose and galactose within their covalent structure.

[0067] In another embodiment, the immunogenic composition comprises cell surface polysaccharides comprising repeating trisaccharide units. In another embodiment, the repeating trisaccharide units comprise GIcNAc, GaINAc, GIcA and optionally, GIu. In another embodiment, the GIcA is non-stoichiometrically substituted with the GIu. In a further embodiment, the GIu is attached to the GIcA through an amide linkage between the 6-carboxyl group of GIcA and the amine of GIu. In another embodiment, the immunogenic composition comprises cell surface polysaccharides further comprising one or more glycerol, glucuronic acid, mannose, glucose and galactose within their covalent structure.

[0068] In another embodiment, the immunogenic composition comprises cell surface polysaccharides comprising repeating trisaccharide units of the formula I and II:

→6-GalNAc-1→4-GlcA-1→3-GlcNAc-1→ (I)

→6-GalNAc-1→4-GlcA-1→3-GlcNAc-1→ (II) 6

1 GIu

[0069] In another embodiment, the immunogenic composition comprises cell surface polysaccharides of the formula PS-1 : [→6)-β-GalNAc-(1→4)-[Glu→6]-β-GlcA-(1→3)-β-GlcNAc-(1]_n wherein n is an integer from 1 to 1000, wherein the 6 carboxyl group of GIc A is optionally substituted with GIu; and immunogenic fragments thereof.

[0070] In one embodiment, n in PS-1 is an integer from 1 to 100, 2 to

100, 10 to 100, 25 to 100, or 5 to 20. [0071] In another embodiment, the immunogenic composition comprising the cell surface polysaccharide comprising the trisaccharide units of the formula I or Il further comprises one or more glycerol, glucuronic acid, mannose, glucose and galactose. [0072] In another embodiment, the immunogenic composition comprising the cell surface polysaccharide of the formula PS-1 further comprises one or more glycerol, glucuronic acid, mannose, glucose and galactose.

[0073] In another embodiment, the immunogenic composition comprises a cell surface polysaccharide (PS-2) comprising phosphate, terminal and 2-substituted glucose, and trisubstituted glycerol within their covalent structure.

[0074] Another aspect of the present application is an immunogenic composition comprising a mixture of: a) the immunogenic composition comprising cell surface polysaccharide PS-1 disclosed herein; and b) the immunogenic composition comprising cell surface polysaccharide PS-2 disclosed herein.

[0075] Another aspect of the present application is an immunogenic composition comprising a mixture of cell surface polysaccharides comprising: a) the cell surface polysaccharide PS-1 disclosed herein; and b) the cell surface polysaccharide PS-2 disclosed herein.

[0076] In another embodiment, the present application discloses a vaccine composition comprising one or more Alloiococcus otitidis cell surface polysaccharides and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or mixtures thereof.

[0077] In an embodiment, the vaccine composition comprises cell surface polysaccharides comprising N-acetyl-D-glucosamine (GIcNAc), N- acetyl-D-galactosamine (GaINAc), D-glucoronic acid (GIcA) and optionally, L- glutamic acid (GIu) within their covalent structure. In another embodiment, the vaccine composition comprises cell surface polysaccharides further comprising one or more glycerol, glucuronic acid, mannose, glucose and galactose within their covalent structure.

[0078] In another embodiment, the vaccine composition comprises cell surface polysaccharides comprising repeating trisaccharide units. In another embodiment, the repeating trisaccharide units comprise GIcNAc, GaINAc₁ GIcA and optionally, GIu. In another embodiment, the GIcA is non- stoichiometrically substituted with the GIu. In a further embodiment, the GIu is attached to the GIcA through an amide linkage between the 6-carboxyl group of GIcA and the amine of GIu. In another embodiment, the vaccine composition comprises cell surface polysaccharides further comprising one or more glycerol, glucuronic acid, mannose, glucose and galactose within their covalent structure.

[0079] In another embodiment, the vaccine composition comprises cell surface polysaccharides comprising repeating trisaccharide units of the formula I and II:

→6-GalNAc-1 →4-GlcA-1 →3-GlcNAc-1 → (I)

→6-GalNAc-1→4-GlcA-1→3-GlcNAc-1→ (II)

6 I

GIu

[0080] In another embodiment, the vaccine composition comprises cell surface polysaccharides of the formula PS-1:

[0081] In one embodiment, n in PS-1 is an integer from 1 to 100, 2 to

100, 10 to 100, 25 to 100, or 5 to 20. [0082] In another embodiment, the vaccine composition comprising the cell surface polysaccharides comprising the trisaccharide units of the formula I or Il further comprises one or more glycerol, glucuronic acid, mannose, glucose and galactose. [0083] In another embodiment, the vaccine composition comprising the cell surface polysaccharides of the formula PS-1 further comprises one or more glycerol, glucuronic acid, mannose, glucose and galactose.

[0084] In another embodiment, the vaccine composition comprises a cell surface polysaccharide (PS-2) comprising phosphate, terminal and 2- substituted glucose, and trisubstituted glycerol within their covalent structure.

[0085] Another aspect of the present application is a vaccine composition comprising a mixture of: a) the vaccine composition comprising cell surface polysaccharide PS-1 disclosed herein; and b) the vaccine composition comprising cell surface polysaccharide PS-2 disclosed herein. [0086] Another aspect of the present application is a vaccine composition comprising a mixture of cell surface polysaccharides comprising: a) the cell surface polysaccharide PS-1 disclosed herein; and b) the cell surface polysaccharide PS-2 disclosed herein.

[0087] Glycoconjugate vaccines are known to enhance the immunogenic properties of carbohydrates. Hence, by coupling isolated cell surface polysaccharides of Alloiococcus otitidis to a carrier molecule, it is possible to maximize the immunogenic response of the carbohydrate-based vaccine.

[0088] Accordingly, in another embodiment of the present application there is included one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein conjugated to one or more carrier molecules.

[0089] In one embodiment, the cell surface polysaccharides comprising

N-acetyl-D-glucosamine (GIcNAc), N-acetyl-D-galactosamine (GaINAc), D- glucoronic acid (GIcA) and optionally, L-glutamic acid (GIu) within their covalent structure are conjugated to one or more carrier molecules. In another embodiment, the cell surface polysaccharides comprising repeating trisaccharide units comprising GIcNAc, GaINAc₁ GIcA and optionally, GIu are conjugated to one or more carrier molecules. In another embodiment, the cell surface polysaccharides comprising repeating trisaccharide units of the formula I or Il are conjugated to one or more carrier molecules. In another embodiment, the cell surface polysaccharides of the formula PS-1 are conjugated to one or more carrier molecules. In another embodiment, the cell surface polysaccharides of the formula PS-2 are conjugated to one or more carrier molecules.

[0090] Another embodiment of the present application is an immunogenic composition comprising one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein conjugated to one or more carrier molecules. [0091] A further embodiment of the present application is a vaccine composition comprising one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein conjugated to one or more carrier molecules.

[0092] In one embodiment, the carrier molecule is a protein. In another embodiment the carrier molecule is bovine serum albumin (BSA). In another embodiment, the carrier molecule is cross reactive material (CRM), for example, CRM₁₉₇. CRM1₉₇ is a nontoxic version of a Diphtheria toxin that has been successfully used in pneumococcal conjugate vaccines (Anderson,

P.W., 1983, Infect. Immun. 39:233-238). In another embodiment, the carrier molecule is MIEP (major immunoenhancing protein). MIEP may be derived from the outer membrane complex of Neisseria meningitis type B and other meningococcal group B (Merck). In another embodiment, the carrier molecule is Diphtheria toxoid. In a further embodiment, the carrier molecule is Tetanus toxoid. In another embodiment, the carrier molecule is a protein derived from Bordetella. [0093] The carrier molecule may be attached to the cell surface polysaccharide using known methods. For example, via an ester or amide linkage between available hydroxy or carboxy groups on the saccharides and carboxyl or amine groups on the protein. [0094] Other carrier molecules and methods of their attachment have been previously reported (see for example US Patent No. 4,673,574, the contents of which are incorporated herein by reference).

[0095] In one embodiment, the cell surface polysaccharide is conjugated to the carrier molecule via carboxylic acid moieties of the GIu and GIcA units of the polysaccharide.

[0096] In one embodiment, the carrier molecule is BSA and is coupled to the Alloiococcus otitidis ceil surface polysaccharide using 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride.

[0097] In one embodiment, the carrier molecule is CRMi₉₇ and is coupled to the Alloiococcus otitidis cell surface polysaccharide using 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0098] In one embodiment, the carrier molecule is Tetanus toxoid and is coupled to the Alloiococcus otitidis cell surface polysaccharide using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. [0099] lmmunogenicity can be significantly improved if the immunizing agent (i.e. the Alloiococcus otitidis cell surface polysaccharide or the immunogenic compositions comprising the Alloiococcus otitidis cell surface polysaccharides or the vaccine compositions comprising the Alloiococcus otitidis cell surface polysaccharides disclosed in the present application) is regardless of administration format, co-immunized with an adjuvant or any other immunostimulatory component. Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic in of themselves. Adjuvants may act by retaining the immunogen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of immunogen to cells of the immune system. Adjuvants can also attract cells of the immune system to an immunogen depot and stimulate such cells to elicit immune response. As such, embodiments of this application encompass pharmaceutical compositions further comprising adjuvants.

[00100] Another aspect of the present application is an immunogenic composition comprising one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein and an immunostimulatory component, for example an adjuvant.

[00101] Another aspect of the present application is a vaccine composition comprising one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein and an immunostimulatory component, for example an adjuvant.

[00102] Adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Intrinsic adjuvants (such as lipopolysaccharides) normally are the components of killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established. [00103] A wide range of extrinsic adjuvants can provoke potent immune responses to immunogens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria and mineral oil, Freund's complete adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes. [00104] In one aspect of this application, adjuvants useful in any of the embodiments described herein are as follows. Adjuvants for parenteral immunization include aluminum compounds (such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate). The antigen can be precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants such as RIBI (ImmunoChem, Hamilton, MT) can also be used in parenteral administration.

[00105] Adjuvants for mucosal immunization include bacterial toxins (e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof). For example, a purified preparation of native cholera toxin subunit B (CTB) can be of use. Fragments, homologs, derivatives, and fusion to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants have been described (e.g., in WO 95/17211 (Arg-7- Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant)). Additional LT mutants that can be used in the methods and compositions of the present application include, for example Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants (such as a bacterial monophosphoryl lipid A (MPLA) of various sources (e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri, saponins, or polylactide glycolide (PLGA) microspheres) can also be used in mucosal administration.

[00106] Adjuvants useful for both mucosal and parenteral immunization include polyphosphazene (for example, WO 95/2415), DC-chol (3 b-(N-(N',N'- dimethyl aminomethane)-carbamoyl) cholesterol (for example, U.S. Patent No. 5,283,185 and WO 96/14831) and QS-21 (for example, WO 88/9336).

[00107] A subject may be immunized with a composition including for example an immunogenic, vaccine or pharmaceutical composition comprising the Alloiococcus otitidis cell surface polysaccharides disclosed in the present application by any conventional route as is known to one skilled in the art. This may include, for example, immunization via a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface, via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route or intranodally. Preferred routes depend upon the choice of the immunogen as will be apparent to one skilled in the art. The administration can be achieved in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by skilled artisans such as the immunogen itself (i.e. peptide vs. nucleic acid (and more specifically type thereof)), the route of administration and the condition of the animal to be vaccinated (weight, age and the like).

[00108] The Alloiococcus otitidis cell surface polysaccharides or immunogenic compositions or vaccine compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., USA, 2000). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

[00109] Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The pharmaceutical composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.

[00110] Compositions including for example immunogenic, vaccine or pharmaceutical compositions of the present application may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA)₁ diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.

[00111] The composition may be in the form of a pharmaceutically acceptable salt which includes, without limitation, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol, histidine, procaine, etc.

[00112] The compositions of the present application can be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration.

[00113] Accordingly, an embodiment of the present application is a pharmaceutical composition comprising an effective amount of one or more Alloiococcus otitidis cell surface polysaccharides of the present application in admixture with a suitable excipient, diluent, carrier, buffer or stabilizer. [00114] In a preferred embodiment, the pharmaceutical compositions are suitable for administration to subjects in a biologically compatible form in vivo.

[00115] Another aspect of the present application is a kit comprising the cell surface polysaccharides disclosed herein or the cell surface polysaccharide mixture disclosed herein or the immunogenic compositions disclosed herein or the vaccine compositions disclosed herein or the pharmaceutical compositions disclosed herein, and instructions for use thereof. [00116] The kit can also include ancillary agents. For example, the kit can include an instrument for injecting the immunogenic composition disclosed herein into a subject, such as a syringe; a vessel for storing or transporting the immunogenic compositions disclosed herein; and/or pharmaceutically acceptable excipients, carriers, buffers or stabilizers. III. METHODS AND USES OF THE APPLICATION

[00117] Another aspect of the present application is a method of inducing an immune response against Alloiococcus otitidis in a subject by administering to the subject an effective amount of one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein. [00118] A further aspect of the present application is a method of treating or preventing Alloiococcus otitidis infection in a subject by administering to the subject an effective amount of one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein.

[00119] An additional aspect of the present application is a method of treating or preventing otitis media in a subject by administering to the subject an effective amount of one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein.

[00120] The present application also includes uses of one or more of the

Alloiococcus otitidis cell surface polysaccharides disclosed herein to induce an immune response against Alloiococcus otitidis in a subject, to treat or prevent Alloiococcus otitidis infection in a subject, and to treat or prevent otitis media in a subject.

[00121] Other aspects of the present application includes uses of one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein for the manufacture of a medicament to induce an immune response against Alloiococcus otitidis in a subject, to treat or prevent Alloiococcus otitidis infection, and to treat or prevent otitis media.

[00122] Another aspect of the present application is a method of inducing an immune response against Alloiococcus otitidis in a subject by administering to the subject an effective amount of the immunogenic compositions disclosed herein where the immunogenic composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein.

[00123] A further aspect of the present application is a method of treating or preventing Alloiococcus otitidis infection in a subject by administering to the subject an effective amount of the immunogenic compositions disclosed herein where the immunogenic composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein. [00124] An additional aspect of the present application is a method of treating or preventing otitis media in a subject by administering to the subject an effective amount of the immunogenic compositions disclosed herein where the immunogenic composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein. [00125] The present application also includes uses of the immunogenic compositions disclosed herein where the immunogenic composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein to induce an immune response against Alloiococcus otitidis in a subject, to treat or prevent Alloiococcus otitidis infection in a subject, and to treat or prevent otitis media in a subject. [00126] Other aspects of the present application includes uses of the immunogenic compositions disclosed herein where the immunogenic composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein for the manufacture of a medicament to induce an immune response against Alloiococcus otitidis in a subject, to treat or prevent Alloiococcus otitidis infection, and to treat or prevent otitis media.

[00127] Another aspect of the present application is a method of inducing an immune response against Alloiococcus otitidis in a subject by administering to the subject an effective amount of the vaccine compositions disclosed herein where the vaccine composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein.

[00128] A further aspect of the present application is a method of treating or preventing Alloiococcus otitidis infection in a subject by administering to the subject an effective amount of the vaccine compositions disclosed herein where the vaccine composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein.

[00129] An additional aspect of the present application is a method of treating or preventing otitis media in a subject by administering to the subject an effective amount of the vaccine compositions disclosed herein where the vaccine composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein.

[00130] The present application also includes uses of the vaccine compositions disclosed herein where the vaccine composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein to induce an immune response against Alloiococcus otitidis in a subject, to treat or prevent Alloiococcus otitidis infection in a subject, and to treat or prevent otitis media in a subject.

[00131] Other aspects of the present application includes uses of the vaccine compositions disclosed herein where the vaccine composition comprises one or more of the Alloiococcus otitidis cell surface polysaccharides disclosed herein for the manufacture of a medicament to induce an immune response against Alloiococcus otitidis in a subject, to treat or prevent Alloiococcus otitidis infection, and to treat or prevent otitis media.

[00132] The methods and uses of the present application are applicable to subjects including for example, pigs, horses, cattle or human beings. In particular, the subject is a human.

[00133] The present application also includes methods and uses of the Alloiococcus otitidis cell surface polysaccharides as diagnostic markers for a Alloiococcus otitidis infection. The sample may be from a human or animal subject or from food or water, or other substance, suspected of infection with Alloiococcus otitidis.

[00134] Accordingly the present application includes of method of detecting Alloiococcus otitidis in a test sample comprising assaying the sample for the presence of one or more of the isolated cell surface polysaccharides disclosed herein. The application also includes the use of one or more of the isolated cell surface polysaccharides disclosed herein to detect Alloiococcus otitidis in a test sample.

[00135] The presence of one of more of the isolated cell surface polysaccharides disclosed herein may be assayed, for example, by isolating the polysaccharides from the sample and performing chemical analyses to determined the identity of the saccharides that are present in the polysaccharide. Such chemical analyses can include one or more of (i) GLC- MS of the corresponding alditol acetates, MS and NMR spectroscopy.

[00136] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[00137] The following non-limiting examples are illustrative of the present invention: Examples Abstract

[00138] Alloiococcus otitidis is a recently discovered Gram-positive bacterium that has been linked with otitis media (middle ear infections). Our aim was to study the covalent chemical structures of putative A. otitidis carbohydrate polymers with the intention of using them in glycoconjugate vaccine preparations. In this application, we describe the structure of a novel cell surface PS expressed by the type-strain of A. otitidis, ATCC 51267, and the synthesis of a glycoconjugate composed of the cell surface PS and bovine serum albumin (BSA). The cell surface PS of A. otitidis type-strain (designated as PS-1) was determined to be a repeating trisaccharide composed of 3-substituted Λ/-acetyl-D-glucosamine (GIcNAc), 6-substituted Λ/-acetyl-D-galactosamine (GaINAc), and 4-substituted D-glucuronic acid (GIcA), of which the majority was amidically decorated with L-glutamic acid (GIu): {→6)-β-GalNAc-(1→4)-[Glu→6]-β-GlcA-(1→3)-β-GlcNAc-(1}_n. Monomeric analysis performed on other A. otitidis strains revealed that similar components were variably expressed, but GIu appeared to be a regular constituent in all strains examined. A second cell surface polysaccharide (designated as PS-2) was determined to comprise phosphate, terminal and 2- substituted glucose, and trisubstituted glycerol within its covalent structure. Due to the suitable presence of GIcA and GIu, our approach for glycoconjugate synthesis employed a carbodiimide-based strategy with activation of available carboxyl groups by 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC), which afforded direct coupling between the cell surface PS and BSA. Analysis by mass- spectrometry indicated that this A. otitidis cell surface PS-BSA conjugate was composed of BSA units that carried up to seven cell surface PSs. This work represeπts the first report in the literature describing an A. otitidis cell-surface polysaccharide and the synthesis of a glycoconjugate preparation thereof.

Materials and Methods

Bacterial growth conditions [00139] A. otitidis type strain ATCC 51267 was isolated from a child with middle ear infection (3), strains SS1337, 1514-89, 1515-89, 1518-89, 1996- 94, 2621-96 were also of similar clinical origin. All strains were obtained from the Centre for Disease Control and Prevention (Atlanta, USA). A. otitidis were grown at 37 ⁰C in a Bacto Todd Hewitt Broth and Tween 80 mixture with relatively slow stirring rate of 60 rpm for 3 days. Cells were harvested by centrifugation at 5000 g at 4 ⁰C for 30 min, and on average, each 4 L batch yielded 2 g of cell paste. The harvested cells were killed by treatment with 1 % phenol and kept in storage at -20 ⁰C.

Extraction and Purification of Cell Surface PS [00140] The lyophilized cells of A. otitidis ATCC 51267 cells were subjected to 2% acetic acid at 100 ⁰C for two hours in order to cleave the putative glycosyl phosphate bridge that links the cell-surface PSs of Gram- positive bacteria to the peptidoglycan layer. The supernatant was dialyzed (1000 amu cut-off), lyophilized and passed through a size exclusion column (1 m x 1 cm) of Bio-Gel P-6 with water as eluent. Carbohydrate containing fractions were detected by the phenol-sulfuric acid assay method (13), and were further scrutinized by 1 -dimensional (1D) nuclear magnetic resonance spectroscopy (NMR) prior to any chemical manipulations. Typically, 1.5 mg of carbohydrate material was obtained from 2 g of cell paste. Sugar Composition Analysis and Linkage Analysis

[00141] Monosaccharide composition analysis was performed by the alditol acetate method (14). The glycosyl hydrolyses were carried out with 4M- trifluoroacetic acid at 105⁰C for 5 hours followed by reduction in H₂O with NaBD₄ overnight at room temperature, or at 80 ⁰C, and subsequent acetylation by acetic anhydride with residual sodium acetate as the catalyst at 100 ⁰C for 2 hours. The alditol acetate derivatives were analyzed by gas liquid chromatography (GLC) using a Varian 3400 gas chromatograph equipped with a 30-m DB-17 capillary column [210⁰C (30 min)-*240°C at 2°C/min], and by GLC-mass spectrometry (GLC-MS) in the electron-impact (El) and chemical-ionization (Cl) modes in a ThermoFinigan PolarisQ instrument. Sugar linkage analysis was performed by the methylation procedure (15) (NaOH/Me2SO/CH₃l) and with characterization of the permethylated alditol acetate derivatives by GLC-MS in the electron impact mode (DB-17 column, isothermally at 190 °C for 60 min). Relative retention times were assigned with respect to glucose (rrt^GIC). Enantiomeric configurations of the individual components were determined by the formation and characterization of the respective 2-(S)- and 2-(R)-butyl chiral glycosides (16).

Mass Spectrometry

[00142] A fraction of the methylated sample was used for analysis by direct electron impact-MS (EI-MS) in the positive mode at 70 eV on an Agilent 6890 GLC-MS instrument coupled to a Micromass/Waters GCT Time-of-Fight mass spectrometer. The methylated sample was dissolved in CH2CI2. The interpretations of the positive ions obtained from EI-MS of the methylated material were similar to those described previously by Dell (17). Electrospray- MS (ES-MS) was obtained with a qTOF ULTIMA GLOBAL instrument (Waters) in the positive ion mode with a capillary (needle) voltage of 1.8 kV, a source temperature of 80 ⁰C, a desolvation gas temperature of 200 ⁰C, and a CID voltage for MS/MS of 30V. The intact material was dissolved in 0.5% acetic acid in acetonitrile/water buffer, whereas the permethylated material was dissolved in a methanol/water/sodium acetate mixture. The Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) MS experiments were carried out in a MALDI Micro MX instrument operated in the linear mode with N2 laser source (337 nm) and positive ion detection. Samples for analysis were mixed with sinapinic acid matrix and 1-2μl were deposited on plate to dry (dry droplet method) and then placed in the spectrometer. Nuclear Magnetic Resonance Spectroscopy

[00143] ¹H and ¹³C NMR spectra were recorded on a Bruker AMX 400 spectrometer at 293 K using standard Bruker software. Prior to performing the NMR experiments, the samples were lyophilized three times with D₂O (99.9%). The HOD peak was used as the internal reference at δ_H 4.821 for ¹H NMR spectroscopy, and orthophosphoric acid (δp 0.0) as external reference for ³¹P NMR experiments. Just before the NMR experiments were carried out, a D2O sample containing TMS (6H 0.00) was run to reference the HOD signal.

Conjugation of Cell Surface PS to Bovine Serum Albumin [00144] The cell surface PS of A. otitidis ATCC 51267 was dissolved in 2-[N-morpholino] ethanesulfonic acid (MES) buffer (pH=5.5), followed by the addition of 1-ethyl-3(3-dimethylamino-propyl) carbodiimide (EDC) in a 1:1 weight ratio to the cell surface PS (adopted from ref. 18). Bovine Serum Albumin (BSA) was immediately added in the same weight ratio and the pH was maintained at 5.5 using MES-acid buffer (0.1 M). The reaction was carried out for 4 hours at room temperature with stirring. The reaction was stopped by raising the pH value to 7 by sodium phosphate buffer (pH=8). The mixture was dialyzed against water for two days. The lyophilized material was analyzed by MALDI-TOF-MS. In this instance, due to the small quantities, no attempt was made to purify the glycoconjugate mixture by size exclusion chromatography. lmmunogenicity Studies

[00145] The polysaccharide was blotted onto a nitrocellulose membrane, and incubated with the supernatants of middle ear effusion specimens from four human patients (#4, #8, #10, #16) with otitis media due to A. otitidis. Then, the membrane was incubated with alkaline phosphatase-conjugated goat anti-human IgG antibody. The blot was visualized with the color substrate, BCIP/NBT. For the Western blot analysis of the polysaccharide, the polysaccharide was electrophoresed in a sodium dodecyl sulfate- polyacrylamide gel, and transferred to a nitrocellulose membrane. The membrane was incubated with the supernatants of middle ear effusion specimens from four human patients (#4, #8, #10, #16) with otitis media due to A. otitidis. Then, the membrane was incubated with alkaline phosphatase- conjugated goat anti-human IgG antibody. The blot was visualized with the color substrate, BCIP/NBT. Monosaccharide Analysis of An Immunogenic Preparation

[00146] Monosaccharide composition analysis of the immunogenic preparation to which IgG, secretory IgA, lgG2, and IgM human antibodies from otitis media patients were detected (11) (Fig. 10) was performed by the alditol acetate method (14). The glycosyl hydrolyses were carried out with 4M- trifluoroacetic acid at 105⁰C for 5 hours followed by reduction in H∑O with NaBD₄ overnight at room temperature, or at 80 ⁰C, and subsequent acetylation by acetic anhydride with residual sodium acetate as the catalyst at 100 ⁰C for 2 hours. The alditol acetate derivatives were analyzed by gas liquid chromatography (GLC) using a Varian 3400 gas chromatograph equipped with a 30-m DB-17 capillary column [21O⁰C (30 min) →240°C at 2°C/min], and by GLC-mass spectrometry (GLC-MS) in the electron-impact (El) and chemical-ionization (Cl) modes in a ThermoFinigan PolarisQ instrument.

Results GLC-MS Analysis of the Water-soluble Extract from A. otitidis ATCC 51267.

[00147] As a starting point in our investigation, we decided to analyze the crude water-soluble preparation that was obtained from the mild-acid treatment of bacterial cells. Monosaccharide composition analysis of the water extract, performed by the characterization of alditol acetate derivatives by GLC-MS (El and positive Cl mode) and with comparison with well defined chemical standards, revealed the presence of Λ/-Acetyl-glucosamine (GIcNAc) and Λ/-Acetyl-galactosamine (GaINAc) as major components, with rrt^GIC=1.33 and rrt^Gl0=1.41 , respectively. Glycerol (Gro) at rrt^GIC=0.19 and GIc (rrt^Glc=1.00) were also observed. Upon analysis in Cl mode it was observed that a portion of the GIc derivative contained two extra atomic mass units (amu), [M+H-60]⁺ = 378 and [M-H]^" = 436, which inferred the presence of glucuronic acid (GIcA) (from the inclusion of two ²H atoms during reduction). In the vicinity of the Gro derivative, two significant peaks at rrt^GIC=0.23 and rrt^GIC=0.35 were determined to be derivatives of glutamic acid (GIu). The monosaccharides mentioned above were determined to possess the D absolute configuration and the GIu residue was confirmed to be present as the L enantiomer.

[00148] Sugar linkage-type analysis carried out by the characterization of the permethylated alditol acetate derivatives (GLC-MS in El mode) showed 3-substituted GIcNAc [→3)-GlcNAc-(1→] and 6-substituted GaINAc [→6)- GaINAc-(I -→] as the dominant units, and, in lower concentrations, tri- substituted Gro, terminal GIc [GIc-(I →], 2-substituted GIc [→2)-Glc-(1→], 4- substituted GIc [→4)-Glc-(1→], and terminal GaINAc [GaINAc-(I →], and GIcNAc [GlcNAc-(1→]. Notably, the 4-substituted GIc mentioned above appeared to emanate from a GIcA residue in that the typical m/z 233, of a 4- substituted permethylated glucitol acetate, was substituted by m/z 235. This preliminary analysis showed that the water-soluble preparation of A. otitidis ATCC 51267 contained a variety of monosaccharide components, Gro, GIc, GIcA, GIcNAc and GaINAc, and one amino acid, GIu, and that GIcNAc and GaINAc appeared to be the main monosaccharide components. GLC-MS Analysis of A. otitidis ATCC 51267 Cell-surface Polysaccharides.

[00149] After mild acetic acid treatment of cells, two main carbohydrate fractions, PS-1 (major) and PS-2 (minor), were obtained by purification of the hydrolysate by size exclusion chromatography. Monosaccharide composition and linkage-type analyses carried out on the fastest eluting fraction (PS-1) revealed that it contained GIcNAc as [→3)-GlcNAc-(1→] and GaINAc as [→6)-GalNAc-(1→]. Small amounts of terminal GaINAc and GIcNAc were also observed to be present in PS-1. Some 4-substituted GIc originating from GIcA, as seen in the previous section, was also detected by GLC-MS here. No other recognizable peaks corresponding to a GIcA permethylated alditol acetate derivative was seen by GLC-MS; however, when the permethylated alditol acetate mixture was analyzed by ESI-MS, an m/z ion at 393.4 was readily observed, which revealed the presence of a di-substituted GIcA unit. Of much less intensity, the mono-substituted GIcA derivative (that which yielded 4-substituted GIc) was also observed at m/z 365.2. Analysis of the monosaccharide derivatives showed that PS-1 was composed of mono- substituted [→3)-GlcNAc-(1→] and [→6)-GalNAc-(1→], and of a GIcA residue, mainly present as a di-substituted residue.

[00150] The second fraction cell surface polysaccharide (PS-2) was composed of phosphate (detected by ³¹P NMR), terminal and 2-substitued GIc, and trisubstitued Gro.

Mass-Spectrometry Analysis of A. otitidis ATCC 51267 Cell Surface PS. [00151] A portion of the intact cell surface PS was analyzed by ESI-MS in combination with a TOF component. The ESI/TOF-MS experiment yielded a variety of multiple charged m/z ions, of which the quadruply charged m/z 1427.08 ion was selected for further MS/MS experiments. Fig. 1 shows the ESI/TOF-MS/MS experiment carried out on m/z 1427.08⁺⁴ ion, which provided m/z ions of defined composition at: m/z 305.96 that represented a GIcA carrying the amino acid GIu; m/z 379.98 for a disaccharide composed of one HexNAc and one GIcA without GIu; m/z 508.98 for a disaccharide composed of one HexNAc and one GIcA(GIu) unit; m/z 582.99 for a trisaccharide containing two HexNAc units and one GIcA residue; m/z 711.99 for a trisaccharide containing two HexNAc units and one GIcA(GIu) residue; m/z 786.52 for a tetrasaccharide composed of three HexNAc units and one GIcA; m/z 914.98 for a tetrasaccharide composed of three HexNAc units and one GIcA(GIu); m/z 1090.99 for a pentasaccharide of three HexNAc units, one GIcA and one GIcA(GIu) residue; m/z 1220.00 for a pentasaccharide composed of three HexNAc units and two GIcA(GIu) residues; and m/z 1423.05 for a hexasaccharide containing four HexNAc residues and two GIcA(GIu) units. To confirm the presence of a GIcA(GIu) residue, an ESI-MS was performed on the fully methylated monomeric units, which were obtained by methylation after acid hydrolysis of the cell surface PS₁ and it showed a m/z at 422.3 corresponding to the MH⁺ of the fully methylated GIcA(GIu). The data obtained from this ESI/TOF-MS/MS experiment afforded structural data indicative of a cell surface PS composed of a trisaccharide with two HexNAc units (GIcNAc and GaINAc as shown by GLC-MS), and one GIcA which was non-stoichiometrically substituted by the amino acid GIu. The relative intensities between ions m/z 379.98 [GaINAc-GIcA] and 508.98 [GaINAc- GIcA(GIu)] suggested that the majority of the GIcA units may be substituted by the GIu moiety.

[00152] In order to obtain monosaccharide sequence information, an El- MS experiment (Fig. 2) was performed on the methylated cell surface PS preparation that yielded m/z ions from the non-reducing end of the cell surface PS. Primary type glycosyloxonium ions, and secondary ions (in brackets) arising from b-elimination (-32 a.m.u.) and double-cleavage (-14 a.m.u.) processes, were observed at m/z 260 (228; 246) for [HexNAc], 505 (473; 491) for [HexNAc→HexNAc], 635 (603; 621) for [HexNAc→GlcA(Glu)], and at 880 (848; 866) for [HexNAc→HexNAc→GlcA(Glu)] or

[HexNAc→GlcA(Glu)→HexNAc], which were indicative of a trisaccharide repeating block composed of two HexNAc units (GaINAc and GIcNAc) and one GIcA(GIu) residue. A secondary ion at m/z 691 (from 723-32) was also observed, which indicated the presence of a trisaccharide repeat at the non- reducing end in which the GIcA residue did not carry a GIu unit [HexNAc→HexNAc-→GlcA→...]. No m/z ions belonging to terminal GIcA {m/z 233) or GIcA(GIu) (m/z 390) were observed, which pointed to the fact that these units did may not present as termini at the non-reducing end of the cell surface PS.

[00153] With the intent of obtaining data pertaining to the molecular weight of the cell surface PS, a portion of intact material was subjected to MALDI-TOF-MS analysis. Two cell surface PS preparations, obtained from two separate growth batches, were analyzed. One cell surface PS preparation, the same used in the ESI/TOF-MS study described above, afforded a high mass m/z ion at 5714, the m/z ion from which the quadruply charged m/z ion 1427.08⁺⁴ (ESI/TOF-MS) originated from. A second cell surface PS preparation (Fig. 3) afforded several m/z ions ranging from 5700s to 6900s with the most intense being observed at m/z 6554. The observed m/z ion at 13175 represents a non-covalent cation-bound dimer species, [2M+H]⁺, which are characteristic artifacts of MALDI-TOF-MS experiments. The MALDI-TOF-MS data indicated that the cell surface PS was composed of approximately 8 (5714 ÷ 712) or 9 (6554 ÷ 712) repeating trisaccharide units fully substituted with GIu: [HexNAc→GlcA(Glu)-1→3-HexNAc]_8-9.

Nuclear Magnetic Resonance Spectroscopy Analysis of A. otitidis ATCC 51267 Ce// Surface PS.

[00154] The ¹H NMR spectrum (Fig. 4) of the cell surface PS showed solely β anomeric proton resonances between dH 4.32 and dH 4.62. No α anomeric resonances were observed. Also readily observed were sharp singlets typical of Λ/-acetyl moieties between dH 2.03 and dH 2.06, and unresolved multiplets characteristic of deoxy protons at dH 1.95 and at dH 2.27. An array of 2D NMR experiments were carried out in order to assign the cell surface PS fine structural features and to re-affirm the previously structural conclusions obtained by MS analysis.

[00155] A 2D ¹H-¹H COSY spectrum (Fig. 5) revealed the presence of five anomeric-related H ₁,₂ cross-peak resonances, which were labeled A, B, C, D and E. The 2D ¹H-¹H COSY spectrum also showed a correlation between d_H 4.17 (W) of a CH unit, and those of methylenes (CH₂) at d_H 2.27 (X),d_H 2.08 (Y) and d_H 1.95 (Z), which revealed that these resonances shared a structural relationship. A total correlation spectroscopy experiment (2D ¹H-¹H TOCSY) was performed, to aid in the assignment of the monosaccharide ring protons, and provided data that readily established the relationship previously observed between the W, X, Y and Z protons, and assigned the resonances of the monosaccharide ring proton resonances which are individually shown in Table ! [00156] A 2D ¹H-¹³C HSQC experiment (Fig. 6A) afforded distinct anomeric carbon resonances at δ_c 99.6 (E), δ_c 100.8 (A)₁ δc 101.0 (D)₁ δc 102.8 (C)₁ and δ_c 103.0 (B). With the aid of the previously described 2D ¹H- ¹H correlation experiments, most of the monosaccharide carbon resonances were assigned (Table 1). The ¹H-¹³C HSQC spectrum also showed carbon signals belonging to the GIcNAc and GaINAc units, with resonances at δc 52.2 (unit D/E) and δ_c 54.4 (unit A), which are characteristic of C-2 of N- Acetyl-hexosamine units, and a broad carbon resonance at δc 21.3 for the N- acetyl moieties. Of particular note, carbon resonances at δc 55.8 (correlating to proton resonance W), δc 34.0 (correlating to proton resonance X), and δc 28.1 (correlating to proton resonances Y and Z), confirmed that these proton resonances emanated from CH (W) and CH₂ (X, Y and Z) structural moieties. Specifically, the proton and carbon resonance of W suggested that the carbon atom in this CH moiety was attached to a nitrogen atom.

[00157] To investigate if there was a nitrogen-carrying moiety in the cell surface PS, a ¹H NMR spectrum was performed in 90% H2O and it showed a distinct NH resonance at 8.63, which was subsequently shown through a ¹H- ¹H COSY a correlation with proton resonance W (dH 4.17). This data revealed that indeed the cell surface PS expressed a CH-NH moiety, and in turn, based on the previous correlations observed, the following moiety could be concluded to be present: HN-CH-CH2-CH₂, the side-chain of the amino acid, GIu.

[00158] In order to gain more concrete information into the monosaccharide sequence, and to observe the carbonyl units of the GIu unit, a ¹H-¹³C HMBC experiment (Fig. 6A and B) was carried out on the cell surface PS. Connectivities (Fig. 6A) were observed between H-1 of B and C- 3 of A (B/C-1→3-A), H-1 of D and C-4 of C (D-1→4-C), H-1 of E and C-4 of B (E-1→4-B), and H-1 of A and C-6 of D and E (A-1→6-D/E):

→6-E-1→4-B-1→3-A→

→6-D-1→4-C-1→3-A→

This experiment showed that the H-1 resonances of both B and C correlated to the C-3 of A. Unit A was thus designated as the 3-substituted GIcNAc to which both B and C could be connected, which implied that B and C was the same unit. The recognition of H-4 and H-5 of the GIcA(GIu) unit, characterized by 2D ¹H-¹³C HMBC (Fig. 6B), belonging to residue B, allowed the assignment of B to GIcA(GIu). In this 2D ¹H-¹³C HMBC₁ no resonances were readily observed for the H-4 and H-5 of GIcA. That a connectivity was seen between H-1 of A [→3)-GlcNAc-(1→] and C-6 of both D and E implied that the 3-substituted GIcNAc was glycosidically attached to the 0-6 position of GaINAc (D/E). H-1 of D and E (6-substituted GaINAc) were in turn connected to the 0-4 of B [GIcA(GIu)] and C (which was assigned to GIcA). The fact that the single 6-substituted GaINAc yielded both D and E anomeric resonances implied that the presence of the variably-substituted GIcA(GIu)* at its C-1 , markedly influenced its chemical shifts. The same was not observed for the 3-substituted GIcNAc (A) to which the GIcA(GIu)* units (B and C) are attached. [00159] Additional spectroscopic information that pointed towards the existence of the linkage sites was obtained by a ¹H-¹H 2D-NOESY (Fig. 7), in that inter-nθe connectivities were observed between H-1 of GIcNAc (A) and H-6 protons of GaINAc (D and E), between H-1 resonances of GIcA(GIu) (B) and GIcA (C) and H-3 of GIcNAc (A), H-1 of GaINAc (D) and H-4 of GIcA (C), and H-1 of GaINAc (E) and H-4 of GIcA(GIu) (B)₁ identifying the cell surface PS trisaccharide repeat:

E B A

→6-GalNAc-1→4-GlcA(Glu)^±-1→3-GlcNAc-→

D C A

[00160] The ¹H-¹³C HMBC spectrum (Fig. 6B) also revealed the presence of three distinct carbonyl moieties. The carbonyl at dc 169.0 correlated to H-4 and H-5 protons of GIcA(GIu) and to the proton resonance

W (CH) of the GIu residue. Another carbonyl at dc 178.0 correlated with protons W, Y and Z, and the third carbonyl at dc 182.0 correlated with protons

X, Y and Z. The association between C-6 (dc 169) of GIcA and the methine proton of GIu illustrated that GIu was amidically linked to the GIcA residue

(shown below):

Synthesis and Characterization of A. otitidis ATCC 51267 Cell Surface PS-BSA Conjugate

[00161] As described above, the cell surface PS of A. otitidis ATCC 51267 expressed carboxylic acids as structural motifs of the GIu and GIcA residues, and thus, we employed these available functional groups in the conjugation to a carrier protein in the design of an anti-A otitidis glycoconjugate vaccine. The cell surface -PS carboxylic acid moieties were transformed to activated carboxylates by EDC, and subsequently the activated capsule PS was directly conjugated to BSA protein to afford an A. otitidis cell surface PS-BSA conjugate. The glycoconjugate preparation was analyzed by MALDI-TOF-MS that revealed the presence of a conjugate with an average molecular weight of 82 KDa, and a molecular weight range that spanned from 66 to 110 KDa (Fig. 8). When taking into account the average molecular weight of the cell surface PS (6554 Da) (Fig. 3) and that of the BSA used here (66349 Da) (determined by MALDI-TOF-MS), one could calculate that a BSA protein carried up to 7 cell surface PSs. The MALDI-TOF-MS spectrum also showed peaks with average molecular weights of 41040 and 164244 KDa, which could be respectively assigned to a doubly charged species and a dimmer species, whose origin could be artificial, as discussed previously. However, there is a possibility that the m/z 164244 ion could indeed represent a covalently linked species carrying 2 BSA units and the corresponding cell surface PSs.

[00162] Parallel conjugation experiments were also carried out using solely GIcA, GIu and also glutamine (GIn) units to compare their conjugation aptitude to BSA using the EDC procedure. The conjugates were analyzed by MALDI-TOF-MS, which showed that the GIcA, GIu and GIn coupled to BSA, and in addition pointed to the fact the GIu (68830 Da) may couple to BSA to a higher degree than GIcA (68359 Da) and GIn (67936 Da).

Monomeric Analysis of other A. otitidis strains. [00163] Monomer composition analyses were performed on the cell surface PSs of strains SS1337 and 1518-89, and on the water-soluble preparations, obtained from mild-acid treatment of cells, of strains 1514-89, 1515-89, 1996-94, and 2621-96. The analyses showed that these strains expressed units similar to those found in the type-strain, namely, GIcNAc₁ GaINAc, GIcA and GIu. Strain 1515-89 was also observed to contain mannose and galactose. Gro and GIc were also detected in the analyses of the water-soluble preparations. Linkage analysis on the cell surface PSs of strains SS1337 and 1518-89 revealed that the GIcNAc and GaINAc units possessed different linkage-types than those found in the type-strain, which indicated that although the above strains contained similar constituents, they need not be arranged in the same manner. This preliminary composition data suggested that these monomeric components may be expressed in several A. otitidis strains, and, more interestingly, that the decoration of cell surface PSs by GIu appears to be ubiquitous. lmmunogenicity of A. otitidis Cell Surface Polysaccharide(s)

[00164] Fig. 9 shows immunological data which demonstrates that the cell surface polysaccharide of A. otitidis is immunogenic in the human middle ear. Fig. 9A shows dot blot analysis of the polysaccharide with clinical specimens. The polysaccharide was blotted onto a nitrocellulose membrane, and incubated with the supernatants of middle ear effusion specimens from four human patients, designated #4, #8, #10 and #16, with otitis media due to A. otitidis. Then, the membrane was incubated with alkaline phosphatase- conjugated goat anti-human IgG antibody. The blot was visualized with the color substrate, BCIP/NBT. The dose-dependent blots revealed that the patients' middle ear effusions carried IgG antibodies specific to the A. otitidis polysaccharide. Fig. 9B shows Western blot analysis of the polysaccharide. The polysaccharide was electrophoresed in a sodium dodecyl sulfate- polyacrylamide gel, and transferred to a nitrocellulose membrane. The membrane was incubated with the supematants of middle ear effusion specimens from four human patients, designated #4, #8, #10 and #16, with otitis media due to A. otitidis. Then, the membrane was incubated with alkaline phosphatase-conjugated goat anti-human IgG antibody. The blot was visualized with the color substrate, BCIP/NBT, which revealed bands in the membranes (arrows) illustrating the fact that otitis media human patients carry specific IgG antibodies in their middle ear effusions to the A. otitidis cell surface polysaccharide. These data suggest the presence of antibody against the cell surface polysaccharide in the middle ear of children with otitis media due to A. otitidis and illustrates that the A. otitidis polysaccharide is immunogenic and has the potential to be an effective antigen in an anti-A otitidis media vaccine. [00165] Fig. 10 shows that an A. otitidis preparation to which IgG, secretory IgA, lgG2, and IgM human antibodies from otitis media patients were detected (11), was composed of a polysaccharide or polysaccharides containing glycerol, glutamic acid, glucuronic acid, mannose, glucose, galactose, N-acetyl-glucosamine and traces of N-acetyl-galactosamine. Discussion

[00166] Otitis media represents one of the major childhood health problems that in addition to grave discomfort can also affect the child's overall learning ability due to hear-loss and speech-delay issues. At the moment there is no vaccine directed solely at fighting otitis media, although Prevnar®, a multivalent glycoconjugate vaccine used primarily in the prevention of invasive pneumococcal disease in infants, is recognized has possessing some ability in reducing otitis media in children (19). One of the main obstacles in curing otitis media is that the origin of this ailment is not always obvious because the causal agent in each situation is difficult to define (20). Here, the cell surface PS of a newly discovered bacterial agent of otitis media, A. otitidis is investigated. In addition to identifying the structure of the cell surface PS, the use of the structurally defined cell surface polysaccharide as a protective immunogen, in the shape of a glycoconjugate vaccine, to combat A. otitidis infections is described.

[00167] The cell surface PS of A. otitidis type-strain ATCC 51267 was determined to be composed of trisaccharide repeating blocks composed of GIcNAc, GaINAc and GIcA, in which the GIcA units were non- stoichiometrically adorned by the amino acid, GIu (structure shown below):

[00168] The majority of GIcA units appeared to carry a GIu residue, as observed by ESI-MS (Fig. 1) experiments and also NMR, in which the anomeric resonance of C (GIcA) was observed to be weaker than that of B [GIcA(GIu)] (Fig. 5). The most striking structural feature of this A. otitidis cell surface PS is the decoration by the amino acid GIu. Inclusions of amino acid units in PSs are not found very frequently, but in fact GIu has been described previously as being a component of PSs produced by Klebsiella [21], Serratia marcescens [22], and Streptomyces fulvissimus [23]. Composition analysis performed on other A. otitidis strains also showed the presence of similar monomeric constituents, which implied that they may be common structural features of numerous A. otitidis strains.

[00169] The design of an anti-A otitidis glycoconjugate vaccine, using the just-described cell surface PS, utilized the carboxylic acid moieties of the

GIu and GIcA units present in the cell surface PS. The cell surface PS-BSA conjugate was synthesized via a carbodiimide-based process and yielded glycoconjugates carrying 1 to 7 cell surface polysaccharides on a BSA protein (Fig. 8). Conjugation experiments using the monomeric constituents, suggested that the carboxylic acids of GIu may be more reactive than those of GIcA. Additionally, since the structural data obtained pointed to the fact that the majority of the GIcA units are substituted by GIu, more carboxylic acids of GIu will be available for conjugation to BSA. Thus, conjugation between the cell surface PS and BSA, in all probability, took place via the carboxylic acids of GIu. The fact that GIn was also observed to couple to BSA implied that both carboxylic acids of GIu could be a participant in conjugation to BSA. Most likely, several carboxylic acid moieties in each cell surface PS were involved in the formation of a link to BSA, which, in all likelihood, yielded a multiple-point attachment conjugate. The average number of carboxylic acids in each cell surface PS involved in attachment to BSA was not calculated here. The apparent regular expression of GIu in the cell surface PSs could be particular useful in the design of an anti-A otitidis multivalent glycoconjugate vaccine, in that it may provide an accessible point of connection between A. otitidis cell surface PSs and carrier protein(s), either by the strategy used here, or through a related coupling approach that exploits the presence of the GIu carboxylic acids. [00170] The minor fraction (PS-2) contained phosphate, Gro and GIc (as terminal and 2-substituted) units.

[00171] An A. otitidis preparation to which IgG, secretory IgA, lgG2, and IgM human antibodies from otitis media patients were detected (11), was observed to be a carbohydrate with an immunogenic composition of glycerol, glutamic acid, glucuronic acid, mannose, glucose, galactose, N-acetyl- glucosamine and N-acetyl-galactosamine (Figure 10).

[00172] The cell surface PS can also be coupled to other carrier proteins that are used in commercially available vaccines (i.e. tetanus toxoid and genetically modified diphtheria toxin, CRMi₉₇). In addition, different designs, such as a single-point attachment conjugate which can be compared immunologically with the multiple-point attachment glycoconjugate described in this work.

[00173] In summary, the newly discovered bacterium, A. otitidis, expresses a cell surface PS, which, due to the presence of GIcA and GIu residues may be considered an acidic cell surface PS. Also, it was shown that a glycoconjugate could be produced in which the cell surface PS may be directly linked to a carrier protein. The use of this glycoconjugate as an anti- A.otitidis vaccine can be tested in a mouse model to evaluate its immunogenicity. The introduction of glycoconjugate vaccines against pneumococcal and influenza infections have shown to be very effective in reducing disease; however, the niche left behind by the respective organisms creates an opening for bacteria such as A. otitidis to prosper, and thus the development of anti-A otitidis vaccine is a priority in the fight against otitis media. [00174] While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00175] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Table 1: ¹H-NMR and ¹³C-NMR chemical shifts (ppm) of monosaccharide units present in A otitidis type-strain ATCC 51267 cell surface PS.

References:

1. American Academy of Family Physicians; American Academy of Otolaryngology-Head and Neck Surgery; American Academy of Pediatrics Subcommittee on Otitis Media With Effusion. (2004) Pediatrics 113, 1412-29.

2. Bluestone, C. D, Stephenson, J. S., and Martin, L. M. (1992) Pediatr. lnfect. Dis. J. ^, Sl-λ λ .

3. Faden, H., and Dryja, D. (1989) J. Clin. Micivbiol. 27, 2488-2491.

4. Aguirre, M., and Collins, M. D. (1992) Int. J. Syst. Bacteriol. 42, 79-83. 5. Gravenitz, A. V. (1993) J. Clin. Microbiol. 31 , 472 (Letter).

6. Hendolin, P. H., Markkanen, A., Ylikoski, J., and Wahlfors, J. J. (1997) J. CHn. Microbiol. 35, 2854-2858.

7. Beswick, A. J., Lawley, B., Fraise, A. P., Pahor, A. L., and Brown, N. L. (1999) Lancet 354, 386-389. 8. Leskinen, K., Hendolin, P., Virolainen-Julkunen, A., Ylikoski, J., and Jero, J. (2002) Int. J. Pediatr. Otorhinolaryngol. 66, 41-48.

9. Harimaya, A., Takada, R., Somekawa, Y., Fujii, N., and Himi, T. (2006) Int. J. Pediatr. Otorhinolaryngol. 70, 1009-1014.

10. Harimaya, A., Takada, R., Hendolin, P. H, Fujii, N., Ylikoski, J., and Himi, T. (2006) J. CHn. Microbiol. 44, 946-949.

11. Harimaya, A., Takada, R., Himi, T., Yokota, S., and Fujii, N. (2007) FEMS Immunol. Med. Microbiol. 49, 41-45.

12. McMichael, J. C, Zagursky, R. J., Fletcher, L. D. (2005) Alloiococcus otitidis open reading frames (orfs) encoding polypeptide antigens, immunogenic compositions and uses thereof, United States Patent

Application 20050203280.

13. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal Chem. 28, 350-356.

14. Sawardeker, J. S., Sloneker, J. H., and Jeanes, A. (1965) Anal. Chem. 37, 1602-1604.

15. Ciucanu, I., and Kerek, F. (1984) Carbohydr. Res. 131, 209-217. 16. Leontein, K., Lindberg, B., and Lonngren, J. (1978) Carbohydr. Res. 62, 359-362.

17. Dell, A. (1987) Adv. Carbohydr. Chem. Biochem. 45, 19-72.

18. Kossaczka, and Szu, S. C. (2000) Glycoconjυgatθ J.. 17, 425-433. 19. Poehling, K. A., Szilagyi, P. G., Grijalva, C. G., Martin, S. W., LaFIeur, B., Mitchel, E., Barth, R. D., Nuorti, J. P., Griffin, M. R. (2007) Pediatrics 119, 707-715.

20. Bhetwal, N., McConaghy, J. R. (2007) Prim. Care. 34, 59-70.

21. Jansson, P. E., Lindberg, B., Widmalm, G., Dutton, G. G., Um, A. V., Sutherland, I. W. (1988) Carbohydr. Res. 175, 103-109.

22. Oxley, D., Wilkinson, S. G. (1990) Carbohydr. Res. 204, 85-91.

23. Shashkov, A. S., Streshinskaya, G. M., Senchenkova, S. N., Kozlova, Y. I., Alferova, I. V., Terekhova, L. P., Evtushenko, L. I. (2006) Carbohydr. Res. 341 , 796-802. 24. Vinogradov, E., Sadovskaya, I., Li, J., and Jabbouri, S. (2006)

Carbohydr. Res. 341 , 738-743. 25. Ying Wang, Y., Johannes Huebner, J., Arthur O. Tzianabos, A. O.,

Gajane Martirosian, G., Dennis L. Kasper, D. L., and Gerald B. Pier, G.

B. (1999) Carbohydr. Res. 316, 155-160.

Footnotes:

The abbreviations used are: BSA, bovine serum albumin; EDC, 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride; El, electron impact; ESI, electrospray ionization; GLC, gas-liquid-chromatography; MALDI-TOF, matrix assisted laser desorption ionization; MS, mass-spectrometry; NMR, nuclear magnetic resonance; PS, polysaccharide; TOF, time of flight.

Claims

Claims:

1. Isolated immunogenic Alloiococcus otitidis cell surface polysaccharides.

2. The cell surface polysaccharides of claim 1, comprising N-acetyl-D- glucosamine (GIcNAc)₁ N-acetyl-D-galactosamine (GaINAc), D-glucoronic acid (GIcA) and optionally, L-glutamic acid (GIu) within their covalent structure.

3. The cell surface polysaccharides of claim 1 or 2 comprising repeating trisaccharide units, the trisaccharide units comprising GIcNAc, GaINAc₁ GIcA and optionally, GIu.

4. The cell surface polysaccharides of any one of claims 2 to 3, wherein the GIcA is non-stoichiometricaily substituted with the GIu.

5. The cell surface polysaccharides of claim 4, wherein the GIu is attached to the GIcA through an amide linkage between the 6-carboxyl group of GIcA and the amine of GIu.

6. The cell surface polysaccharides of any one of claims 1 to 5, comprising the repeating trisaccharide units of the formula (I) and (II):

→6-GalNAc- 1 -→4-GlcA-1 →3-GlcNAc-1 → (I)

→6-GalNAc-1→4-GlcA-1→3-GlcNAc-1→ (II)

6 1 GIu

7. The cell surface polysaccharides of any one of claims 2 to 6, wherein the polysaccharide is of the formula PS-1:

[→6)-β-GalNAc-(1 →4)-β-GlcA-(1 →3)-β-GlcNAc-(1 ]_n wherein n is an integer from 1-1000, wherein the 6 carboxyl group of GIc A is optionally substituted with GIu; and immunogenic fragments thereof.

8. The cell surface polysaccharides of claim 7, wherein n is an integer from 1 to 100.

9. The cell surface polysaccharides of claim 6, wherein n is an integer from 5 to 20.

10. The cell surface polysaccharides of any one of claims 1 to 9, wherein their covalent structure further comprises one or more glycerol, glucuronic acid, mannose, glucose and galactose.

11. The cell surface polysaccharides of claim 1 isolated from A. otitidis cells by treatment of the cells with mild acid under conditions to cleave cell surface polysaccharides from the cells.

12. The cell surface polysaccharides of claim 11, wherein the mild acid is 0.1 % to 5% acetic acid.

13. The cell surface polysaccharides of claim 11 or 12, wherein the polysaccharides are further purified by dialysis, lyophilization and/or size exclusion chromatography.

14. The cell surface polysaccharides of any one of claims 1 and 11 to 13, wherein the polysaccharides comprise phosphate, terminal and 2-substituted glucose, and trisubstituted glycerol within their covalent structure.

15. The cell surface polysaccharides of any one of claims 1 to 14, wherein the polysaccharides are conjugated to one or more carrier molecules.

16. The cell surface polysaccharides of claim 15, wherein said carrier molecule is BSA, CRM1₉7, MIEP, Dipthteria toxoid, Tetanus toxoid or proteins derived from Bordetella.

17. The cell surface polysaccharides of claim 15 or 16, wherein the polysaccharides are conjugated to the carrier molecule via carboxylic acid moieties of the GIu and GIcA units of the polysaccharide.

18. The cell surface polysaccharides of claim 16 or 17, wherein said carrier molecule is BSA and the BSA is coupled to the polysaccharide using 1-ethyl-

3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

19. The cell surface polysaccharides of claim 16 or 17, wherein said carrier molecule is CRMi₉₇ and the CRMig₇ is coupled to the polysaccharide using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

20. The cell surface polysaccharides of claim 16 or 17, wherein said carrier molecule is Tetanus toxoid and the Tetanus toxoid is coupled to the polysaccharide using 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

21. A cell surface polysaccharide mixture comprising the polysaccharide as defined in any one of claims 1 to 13 and the polysaccharide as defined in claim 14.

22. The cell surface polysaccharide mixture of claim 21 , wherein one or more of the polysaccharides are conjugated to a carrier molecule.

23. The cell surface polysaccharide of claim 22, wherein said carrier molecule is BSA₁ CRIVW, MIEP, Dipthteria toxoid, Tetanus toxoid or proteins derived from Bordetella.

24. The cell surface polysaccharide of claim 22 or 23, wherein the polysaccharide is conjugated to the carrier molecule via carboxylic acid moieties of the GIu and GIcA units of the polysaccharide.

25. The cell surface polysaccharide of claim 22, wherein said carrier molecule is BSA and the BSA is coupled to the polysaccharide using 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

26. The cell surface polysaccharide of claim 22, wherein said carrier molecule is CRMi₉₇ and the CRIvW is coupled to the polysaccharide using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

27. The cell surface polysaccharide of claim 22, wherein said carrier molecule is Tetanus toxoid and the Tetanus toxoid is coupled to the polysaccharide using 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

28. An immunogenic composition comprising the cell surface polysaccharide of any one of claims 1 to 20 and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or mixtures thereof.

29. An immunogenic composition mixture comprising the cell surface polysaccharide mixture of any one of claims 21 to 27 and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or mixtures thereof.

30. The immunogenic composition of claim 28 or 29, further comprising an immunostimulatory component, such as an adjuvant.

31. A vaccine composition comprising the cell surface polysaccharide of any one of claims 1 to 20 and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or mixtures thereof.

32. A vaccine composition mixture comprising the cell surface polysaccharide mixture of any one of claims 21 to 27 and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or mixtures thereof.

33. The vaccine composition of claim 31 or 32, further comprising an immunostimulatory component, such as an adjuvant.

34. A kit comprising the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27 or the immunogenic composition of claims 28 to 30 or the vaccine composition of any one of claims 31 to 33 and instructions for the use thereof.

35. A method of inducing an immune response against Alloiococcus otitidis in a subject, comprising administering to said subject an effective amount of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27.

36. A method of treating or preventing Alloiococcus otitidis infection in a subject, comprising administering to said subject an effective amount of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27.

37. A method of treating or preventing otitis media in a subject, comprising administering to said subject an effective amount of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27.

38. A method of inducing an immune response against Alloiococcus otitidis in a subject, comprising administering to said subject an effective amount of the immunogenic composition of any one of claims 28 to 30.

39. A method of treating or preventing Alloiococcus otitidis infection in a subject, comprising administering to said subject an effective amount of the immunogenic composition of any one of claims 28 to 30.

40. A method of treating or preventing otitis media in a subject, comprising administering to said subject an effective amount of the immunogenic composition of any one of claims 28 to 30.

41. A method of inducing an immune response against Alloiococcus otitidis in a subject, comprising administering to said subject an effective amount of the vaccine composition of any one of claims 31 to 33.

42. A method of treating or preventing Alloiococcus otitidis infection in a subject, comprising administering to said subject an effective amount of the vaccine composition of any one of claims 31 to 33.

43. A method of treating or preventing otitis media in a subject, comprising administering to said subject an effective amount of the vaccine composition of any one of claims 31 to 33.

44. Use of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27 to induce an immune response against Alloiococcus otitidis in a subject.

45. Use of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27 for the manufacture of a medicament to induce an immune response against Alloiococcus otitidis in a subject.

46. Use of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27 for treating or preventing Alloiococcus otitidis infection in a subject.

47. Use of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27 for the manufacture of a medicament for treating or preventing Alloiococcus otitidis infection in a subject.

48. Use of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27 for treating or preventing otitis media in a subject.

49. Use of the cell surface polysaccharide of any one of claims 1 to 20 or the cell surface polysaccharide mixture of any one of claims 21 to 27 for the manufacture of a medicament for treating or preventing otitis media in a subject.

50. Use of the immunogenic composition of any one of claims 28 to 30 to induce an immune response against Alloiococcus otitidis in a subject.

51. Use of the immunogenic composition of any one of claims 28 to 30 for the manufacture of a medicament to induce an immune response against Alloiococcus otitidis in a subject.

52. Use of the immunogenic composition of any one of claims 28 to 30 for treating or preventing Alloiococcus otitidis infection in a subject.

53. Use of the immunogenic composition of any one of claims 28 to 30 for the manufacture of a medicament for treating or preventing Alloiococcus otitidis infection in a subject.

54. Use of the immunogenic composition of any one of claims 28 to 30 for treating or preventing otitis media in a subject.

55. Use of the immunogenic composition of any one of claims 28 to 30 for the manufacture of a medicament for treating or preventing otitis media in a subject.

56. Use of the vaccine composition of any one of claims 31 to 33 to induce an immune response against Alloiococcus otitidis in a subject.

57. Use of the vaccine composition of any one of claims 31 to 33 for the manufacture of a medicament to induce an immune response against Alloiococcus otitidis in a subject.

58. Use of the vaccine composition of any one of claims 31 to 33 for treating or preventing Alloiococcus otitidis infection in a subject.

59. Use of the vaccine composition of any one of claims 31 to 33 for the manufacture of a medicament for treating or preventing Alloiococcus otitidis infection in a subject.

60. Use of the vaccine composition of any one of claims 31 to 33 for treating or preventing otitis media in a subject.

61. Use of the vaccine composition of any one of claims 31 to 33 for the manufacture of a medicament for treating or preventing otitis media in a subject.

62. The method of any one of claims 35 to 43, wherein the subject is a human being.

63. The use according to any one of claims 44 to 61 , wherein the subject is a human being.