WO2002089839A1 - Immunisation intranasale par un lipooligosaccharide detoxifie provenant d'un haemophilus influenzae ou d'un moraxella catarrhalis non typable - Google Patents

Immunisation intranasale par un lipooligosaccharide detoxifie provenant d'un haemophilus influenzae ou d'un moraxella catarrhalis non typable Download PDF

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WO2002089839A1
WO2002089839A1 PCT/US2001/032331 US0132331W WO02089839A1 WO 2002089839 A1 WO2002089839 A1 WO 2002089839A1 US 0132331 W US0132331 W US 0132331W WO 02089839 A1 WO02089839 A1 WO 02089839A1
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Prior art keywords
aerosolizer
delivery system
dlos
system comprises
mucosal adjuvant
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PCT/US2001/032331
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English (en)
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Xin-Xing Gu
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Priority to CA002446355A priority Critical patent/CA2446355A1/fr
Publication of WO2002089839A1 publication Critical patent/WO2002089839A1/fr
Priority to US10/688,115 priority patent/US20040126381A1/en
Priority to US11/260,773 priority patent/US7641906B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • A61K39/1045—Moraxella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

  • the invention relates to intranasal immunization with detoxified lipooligosaccharide from nontypeable Haemophilus in ⁇ uenzae or Moraxella catarrhalis.
  • Nontypeable Haemophilus influenzae is an important cause of otitis media (OM) in children and respiratory tract diseases in adults (Klein, J.O. et al. 1992 Adv Pediatr 39:127-156; Murphy, T.F. et al. 1987 Rev Infect Dis 9:1-15; Musher, D.M. et al. 1983 Ann Intern Med 99:344-350).
  • Moraxella Branhamella
  • catarrhalis Catlin, B.W. 1990 Clin Microbiol Rev 3:293-320; Doern, G.V. 1986 Diagn Microbiol Infect Dis 4:191-201; Enright, M.C., and H.
  • Nontypeable Haemophilus influenzae is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance.
  • Lipooligosaccharide LOS
  • dLOS detoxified LOS
  • dLOS was bound to tetanus toxoid (TT) or high-molecular-weight proteins ( ⁇ MPs) from NT ⁇ i through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS- ⁇ MP.
  • TT tetanus toxoid
  • ⁇ MPs high-molecular-weight proteins
  • the molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1.
  • the antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion.
  • Moraxella (Branhamella) catarrhalis (M. catarrhalis) is an important cause of otitis media and sinusitis in children and of lower respiratory tract infections in adults.
  • Lipooligosaccharide (LOS) is a major surface antigen of the bacterium and elicits bactericidal antibodies.
  • Treatment of the LOS from strain ATCC 25238 with anhydrous hydrazine reduced its toxicity 20,000-fold, as assayed in the Limulus amebocyte lysate (LAL) test.
  • the detoxified LOS was coupled to tetanus toxoid (TT) or high- molecular-weight proteins (HMP) from nontypeable Haemophilus influenzae through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP.
  • TT tetanus toxoid
  • HMP high- molecular-weight proteins
  • the molar ratios of dLOS to TT and HMP conjugates were 19:1 and 31:1, respectively.
  • the antigenicity of the two conjugates was similar to that of the LOS, as determined by double immunodiffusion.
  • the invention relates to intranasal immunization with detoxified lipooligosaccharide from nontypeable Haemophilus influenzae ox Moraxella catarrhalis. Brief Description of the Drawings
  • Figure A shows the proposed chemical structure of lipid A from nontypeable
  • Figure B shows the proposed chemical structure of lipid A from Moraxella catarrhalis lipooligosaccharide (LOS).
  • R site of attachment of the oligosaccharide chain.
  • Hydrazine treatment of LOS removes primary O-linked fatty acids from 3-hydroxy groups of diglucosamine (*) and secondary O-linked fatty acids from hydroxy groups of 3-hydroxy fatty acids of lipid A (arrow).
  • FIG. 1 Immunohistochemistry with anti-IgA and anti-IgG staining in the nose.
  • A Anti-IgA (or anti-IgG) staining in control mice,
  • B anti-IgA staining in dLOS-TT immunized mice, and
  • C anti-IgG staining in dLOS-TT immunized mice (magnification 400x).
  • Intranasal immunization with dLOS-TT dramatically increased the staining with IgA of the mucous blanket, and glandular cells in the nose as compared with the staining in the control mice.
  • staining with anti-IgG was strongly shown only at the vessels of the nasal tissue in mice immunized with dLOS-TT.
  • the nasal tissue of the control mice was not stained with anti-IgA (or anti-IgG).
  • FIG. 1 Bacterial clearance of NTHi strain 9274 from mouse nasopharynx. Immunization schedules and mouse grouping were shown in Table 1, footnote a. Mice were challenged with strain 9274 into the nose 1 wk after the last immunization and nasal washes were collected at 6 h post-challenge. Mice immunized with dLOS-TT and CT showed a significant reduction of bacterial recovery by 74% or 76% when compared to those of the mice immunized with CT alone or dLOS and CT (*, p ⁇ 0.05).
  • FIG. 3 Binding reactivity of nasal wash (IgA) or serum (IgG) to homologous strain and five heterologous strains in whole-cell ELISA.
  • the nasal wash from mice immunized with dLOS-TT bound strongly to the homologous strain 9274 and the heterologous strains 3198, 5657 and 7502 but weakly to strains 1479 and 2019 (A). Similar binding reactivity was observed in serum from mice immunized with the dLOS-TT (B).
  • FIG. 4 Silver-stained SDS-PAGE patterns (A) and Western blot analysis (B and C) of homologous strain and five heterologous strains. Lanes 1 through 6 contain strains 1479, 2019, 3198, 5657, 7502 and 9274. Nasal wash (IgA) from mice immunized with dLOS-TT was reactive strongly to LOSs from strains 9274, 3198, 5657, and 7502, weakly to 1479 but not to 2019 (B). However, sera (IgG) from mice immunized with the dLOS-TT were reactive to all LOSs with strong binding in strains 9274, 3198, 5657 and 7502 (C). Arrows show each LPS of Ra (upper arrow) and Re (lower arrow) mutants as markers from Salmonella minnesota.
  • Figure I Specific antibody-forming cells induced by dLOS-CRM conjugate measured by ELISPOT assay. See Table I, footnote a.
  • A IgA-forming cells per million of lymphoid cells
  • B IgG-forming cells per million of lymphoid cells
  • C IgM-forming cells per million of lymphoid cells.
  • NALT nasal-associated lymphoid tissue
  • NP nasal passage
  • CLN cervical lymph node
  • PP Peyer's patch.
  • Figure II Specific antibody-forming cells initiated by different dLOS-protein conjugates. See Table III, footnote a. NALT: nasal-associated lymphoid tissue, NP: nasal passage, CLN: cervical lymph node, PP: Peyer's patch.
  • Figure III Comparison of protective effect induced by different dLOS-protein conjugates in bacterial clearance from mouse nasopharynx and lungs. See Table III, footnote a.
  • mice were challenged with 2 x 10 8 CFU of M. catarrhalis strain 25238 per ml in a nebulizer, and nasal washes and lungs were collected at 6 h postchallenge.
  • the CFU of bacterial recovery from CT group compared to that of other group: P ⁇ 0.01.
  • Figure IV Comparison of protective effect from different immunization regimens in bacterial clearance from mouse nasopharynx. See Table IV, footnote a.
  • mice were challenged with 2 x 10 s CFU of M. catarrhalis strain 25238 per ml in a nebulizer, and nasal washes were collected at 6 h postchallenge. Green bars: intranasal immunization, red bars: subcutaneous injection.
  • Figure V Comparison of protective effect from different immunization regimens in bacterial clearance from mouse lungs. See Table IV, footnote a.
  • mice were challenged with 2 x 10 s CFU of M. catarrhalis strain 25238 per ml in a nebulizer, and lungs were collected at 6 h postchallenge. Green bars: intranasal immunization, red bars: subcutaneous injection.
  • Figure VI Kinetics of bacterial recovery from mouse nasopharynx challenged with
  • M. catarrhalis strain 25238 Mice were intranasally administered 4 times at 1-week intervals with 10 ⁇ l of PBS containing a mixture of 5 ⁇ g of dLOS -CRM and 1 ⁇ g of CT, or 10 ⁇ l of PBS.
  • immunized mice significantly reduced bacterial recovery from nasopharynx and lungs, and bacterial recovery became undetectable within 24 h, postchallenge.
  • the invention relates to an immunogenic composition
  • an immunogenic composition comprising an immunizing amount of Nontypeable Haemophilus influenzae (NTHi) or Moraxella catarrhalis lipooligosaccharide (LOS) from which at least one primary O-linked fatty acid has been removed to form detoxified LOS (dLOS) and an immunogenic carrier covalently linked thereto, optionally where the dLOS and the immunogenic carrier are covalently linked by a linker, and a mucosal adjuvant or delivery system.
  • NHi Nontypeable Haemophilus influenzae
  • LOS Moraxella catarrhalis lipooligosaccharide
  • dLOS detoxified LOS
  • an immunogenic carrier covalently linked thereto, optionally where the dLOS and the immunogenic carrier are covalently linked by a linker, and a mucosal adjuvant or delivery system.
  • catarrhalis lipooligosaccharide from which at least one primary O-linked fatty acid has been removed to form detoxified LOS (dLOS) and an immunogenic carrier covalently linked thereto, optionally where the dLOS and the immunogenic carrier are covalently linked by a linker, elicits an immunological response and can even inhibit colonization by NTHi or M. catarrhalis and prevent otitis media and other respiratory diseases caused by NTHi or M. catarrhalis infection.
  • the present invention provides a method for inducing an immunological response in a host, preferably a human host, to inhibit colonization by NTHi or M. catarrhalis or prevent otitis media and other respiratory diseases caused by NTHi or M. catarrhalis infection by mucosal administration, preferably intranasal administration, to the host of an effective amount of NTHi or M.
  • LOS catarrhalis lipooligosaccharide
  • dLOS detoxified LOS
  • immunogenic carrier covalently linked thereto, optionally where the dLOS and the immunogenic carrier are covalently linked by a linker, and a mucosal adjuvant or delivery system.
  • the present invention provides use of an effective amount of NTHi or M. catarrhalis lipooligosaccharide (LOS) from which at least one primary O-linked fatty acid has been removed to form detoxified LOS (dLOS) and an immunogenic carrier covalently linked thereto, optionally where the dLOS and the immunogenic carrier are covalently linked by a linker, and a mucosal adjuvant or delivery system, for mucosal administration, preferably intranasal administration, to a host, preferably a human host, for inducing an immunological response to inhibit colonization by NTHi or M. catarrhalis or prevent otitis media and other respiratory diseases caused by NTHi or catarrhalis infection.
  • LOS NTHi or M. catarrhalis lipooligosaccharide
  • dLOS detoxified LOS
  • a mucosal adjuvant or delivery system for mucosal administration, preferably intranasal administration, to a host, preferably a human
  • the present invention relates to a conjugate vaccine comprising nontypeable Haemophilus influenzae (NTHi) or Moraxella catarrhalis lipooligosaccharide (LOS) from which at least one primary O-linked esterified fatty acid has been removed to form detoxified LOS (dLOS), and an immunogenic carrier covalently linked thereto, optionally where the dLOS and immunogenic carrier are covalently linked by a linker.
  • LOS may be extracted from NTHi or M. catarrhalis and purified according to conventional processes.
  • NTHi and M. catarrhalis lipooligosaccharides may be of any serotype.
  • serotypes I, II, III, IV and V for NTHi are cited (Campagnari, A. A. et al. 1987 Infect Immun 55:882-887; Partick, C.C. et al. 1987 Infect Immun 55:2902-2911), but the LOS used for the conjugates herein was highly cross-reactive to the majority of NTHi clinical isolates.
  • M. catarrhalis three major LOS serotypes: A, B and C are cited (Vaneechoutte, M.G. et al. 1990 J Clin Microbiol 28:182-187).
  • One or several lipooligosaccharides may be concomitanfiy administered by the mucosal route.
  • the medicament, i.e., the vaccine, for mucosal administration may contain several lipooligosaccharides, each of a particular serotype.
  • Fig. A A proposed chemical structure of lipid A from nontypeable Haemophilus influenzae lipooligosaccharide (LOS) is shown in Fig. A.
  • Fig. B A proposed chemical structure of lipid A from Moraxella catarrhalis lipooligosaccharide (LOS) is shown in Fig. B.
  • the O-linked esterified fatty acids shown by the asterisks are defined as primary O-linked fatty acids and those shown by the arrows are defined as secondary O-linked fatty acids.
  • the conjugate vaccine may also comprise LOS from which both primary O-linked fatty acids have been removed. In addition to the removal of at least one primary O-linked fatty acid from LOS, one or both of the secondary O-linked fatty acids may also be removed.
  • the number of primary and secondary O-linked fatty acids removed by hydrazine treatment, or by treatment with any other reagent capable of hydrolyzing these linkages, will depend on the time and temperature of the hydrolysis reaction.
  • the determination of the number of fatty acid chains which have been removed during the reaction can be determined by standard analytical methods including mass spectrometry and nuclear magnetic resonance (NMR).
  • hydrazine for detoxification of LOS from NTHi or M. catarrhalis
  • any reagent or enzyme capable of removing at least one primary O-linked fatty acid from LOS is within the scope of the present invention.
  • other bases such as sodium hydroxide, potassium hydroxide, and the like may be used.
  • dLOS is optionally conjugated to a linker, such as adipic acid dihydrazide (ADH), prior to conjugation to an immunogenic carrier protein, such as tetanus toxoid (TT).
  • ADH adipic acid dihydrazide
  • TT tetanus toxoid
  • dLOS may be directly covalently bonded to the carrier. This may be accomplished, for example, by using the cross-linking reagent glutaraldehyde. However, in a preferred embodiment, dLOS and the carrier are separated by a linker.
  • linker promotes optimum immunogenicity of the conjugate and more efficient coupling of the dLOS with the carrier.
  • Linkers separate the two antigenic components by chains whose length and flexibility can be adjusted as desired. Between the bifunctional sites, the chains can contain a variety of structural features, including heteroatoms and cleavage sites. Linkers also permit corresponding increases in translational and rotational characteristics of the antigens, increasing access of the binding sites to soluble antibodies.
  • suitable linkers include, for example, heterodifunctional linkers such as ⁇ -aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenyl amine.
  • Coupling reagents contemplated for use in the present invention include hydroxysuccinimides and carbodumides. Many other linkers and coupling reagents known to those of ordinary skill in the art are also suitable for use in the invention (e.g. cystamine). Such compounds are discussed in detail by Dick et al. (Dick et al. Conjugate Vaccines, J.M. Cruse and R.E. Lewis, Jr., eds. Karger, New York, pp. 48-114, 1989).
  • Polymeric immunogenic carriers can be a natural or synthetic material containing a primary and/or secondary amino group, an azido group or a carboxyl group.
  • the carrier may be water soluble or insoluble.
  • immunogenic carrier proteins Any one of a variety of immunogenic carrier proteins may be used in the conjugate vaccine of the present invention.
  • classes of proteins include pili, outer membrane proteins and excreted toxins of pathogenic bacteria, nontoxic or "toxoid” forms of such excreted toxins, nontoxic proteins antigenically similar to bacterial toxins (cross-reacting materials or CRMs) and other proteins.
  • Nonlimiting examples of bacterial toxoids contemplated for use in the present invention include tetanus toxin/toxoid, diphtheria toxin toxoid, detoxified P.
  • aeruginosa toxin A cholera toxin/toxoid, pertussis toxin/toxoid and Clostridium perfringens exotoxins/toxoid.
  • the toxoid forms of these bacterial toxins are preferred.
  • viral proteins i.e. hepatitis B surface/core antigens; rotavirus VP 7 protein and respiratory syncytial virus F and G proteins
  • CRMs include CRM197, antigenically equivalent to diphtheria toxin (Pappenheimer et al. 1972 Immunochem 9:891-906) and CRM3201, a genetically manipulated variant of pertussis toxin (Black et al. 1988 Science 240:656-659).
  • immunogenic carrier proteins from non-mammalian sources including keyhole limpet hemocyanin, horseshoe crab hemocyanm and plant edestin is also within the scope of the invention.
  • Outer membrane proteins include high molecular weight proteins (HMPs), P4 and P6 from nontypeable Haemophilus influenzae and CD and USPA from Moraxella catarrhalis.
  • HMPs high molecular weight proteins
  • P4 and P6 from nontypeable Haemophilus influenzae
  • CD and USPA from Moraxella catarrhalis.
  • PCT WO98/53851 There are many coupling methods which can be envisioned for dLOS-protein conjugates.
  • dLOS is selectively activated by l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC)-mediated ADH derivatization of the terminal 3- deoxy-D-manno-2-octulosonic acid (KDO) group of dLOS, followed by EDC-mediated coupling to TT.
  • EDC l-ethyl-3-(3- dimethylaminopropyl) carbodiimide
  • KDO deoxy-D-manno
  • cystamine derivatization of dLOS by, for example, EDC-mediated derivatization, followed by disulfide conjugation to N-succimidyl-3-(2-pyridyldithio) propionate- derivatized protein.
  • EDC-mediated derivatization followed by disulfide conjugation to N-succimidyl-3-(2-pyridyldithio) propionate- derivatized protein.
  • Other methods well known in the art for effecting conjugation of oligosaccharides to immunogenic carrier proteins are also within the scope of the invention. Such methods are described in, for example, U.S. Patent Nos. 5,153,312 and 5,204,098; and EP 0 497 525; and EP 0 245 045.
  • the molar ratio of ADH to dLOS in the reaction mixture is typically between about 10:1 and about 250:1.
  • a molar excess of ADH is used to ensure more efficient coupling and to limit dLOS-dLOS coupling.
  • the molar ratio is between about 50:1 and about 150:1; in a most preferred embodiment, the molar ratio is about 100:1.
  • Similar ratios of AH-dLOS to both TT and HMP in the reaction mixture are also contemplated, hi a preferred embodiment, one ADH per dLOS is present in the AH-dLOS conjugate, hi another preferred embodiment, in the final dLOS-carrier protein conjugate, the molar ratio of dLOS to carrier is between about 15 and about 75, preferably between about 25 and about 50.
  • Immunogenic compositions including vaccines may be prepared as inhalables, sprays and the like (e.g., nasal spray, aerosol spray or pump spray and the like), e.g., as liquid solutions or emulsions, etc.
  • Aerosol spray preparations can be in a pressurized container with a suitable propellant such as a hydrocarbon propellant.
  • Pump spray dispensers can dispense a metered dose or, a dose having a particular particle or droplet size. Pump spray dispensers are commercially available, e.g., from Valois of America, Inc., Connecticut.
  • Nasal spray dispensers are commonly fabricated from a flexible material such as plastic and cause a spray to dispense in response to being squeezed.
  • Anti-inflammatories such as "Vanceril” are commercially available in oral and nasal aerosol form for mucosal administration; the anti-inflammatory "Vancerase” is commercially available in a pump- spray dispenser for nasal administration; cold remedies such as “Dristan” are commercially available in nasal spray (squeeze) dispensers (so that the reader is aware that aerosol, pump and squeeze dispensers are known and available).
  • the lipooligosaccharide may be mixed with pharmaceutically acceptable excipients which are compatible therewith.
  • excipients may include water, saline, dextrose, glycerol, ethanol, and combinations thereof.
  • the immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or mucosal adjuvants or delivery systems to enhance the effectiveness thereof.
  • the lipooligosaccharide is combined with a mucosal adjuvant or delivery system.
  • a mucosal adjuvant or delivery system See Singh, M. & O'Hagan, D., Nov 1999 Nature Biotechnology 17:1075-1081; and Ryan, E.J. et al. Aug 2001 Trends in Biotechnology 19:293-304.
  • Suitable mucosal adjuvants and delivery systems are listed in the table below. Table: Mucosal Adjuvants and Delivery Systems
  • Cytokines e.g., IL-1, IL-2, IL-12, IFN- ⁇ , GM-CSF
  • LPS Lipopolysaccharide
  • Emulsions e.g., Freund's, SAF, MF59
  • PLG Poly(lactide-co-glycolides)
  • CT Cholera toxin
  • Mutant toxins e.g., LTK63 and LTR72
  • the mucosal administration preferably is effected intranasally, e.g., to the olfactory mucosa, to provide protection to the host against both bacterial colonization and systemic infection.
  • the intranasal administration also may provide protection to the host against pulmonary infection as well as protection to the host against an infection starting as a pulmonary infection.
  • the mucosal administration can also involve respiratory mucosa, gingival mucosa or alveolar mucosa.
  • the administration can be perlingual or sublingual or into the mouth or respiratory tract; but intranasal administration is preferred.
  • compositions of the invention are conveniently provided as isotonic aqueous solutions, suspensions or viscous compositions which may be buffered to a selected pH.
  • the viscous compositions may be in the form of gels, lotions, ointments, creams and the like and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2500 to 6500 cps, although more viscous compositions, even up to 10,000 cps may be employed.
  • Viscous compositions have a viscosity preferably of 2500 to 5000 cps, since above that range they become more difficult to administer.
  • Liquid sprays and drops are normally easier to prepare than gels and other viscous compositions. Additionally, they are somewhat more convenient to administer, especially in multi-dose situations. Viscous compositions, on the other hand can be fonnulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the nasal mucosa.
  • Suitable nontoxic pharmaceutically acceptable carriers, and especially nasal carriers will be apparent to those skilled in the art of pharmaceutical and especially nasal pharmaceutical formulations. For those not skilled in the art, reference is made to the text entitled Remington 's Pharmaceutical Sciences, a reference book in the field. Obviously, the choice of suitable carriers will depend on the exact nature of the particular mucosal dosage form, e.g., nasal dosage form, required [e.g., whether the composition is to be formulated into a solution such as a nasal solution (for use as drops or as a spray), a nasal suspension, a nasal ointment, a nasal gel or another nasal form].
  • Preferred mucosal and especially nasal dosage forms are solutions, suspensions and gels, which normally contain a major amount of water (preferably purified water) in addition to the antigen (PspA). Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents and jelling agents (e.g., methylcellulose) may also be present.
  • pH adjusters e.g., a base such as NaOH
  • emulsifiers or dispersing agents e.g., a base such as NaOH
  • buffering agents e.g., preservatives
  • wetting agents and jelling agents e.g., methylcellulose
  • jelling agents e.g., methylcellulose
  • compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • sodium chloride is preferred particularly for buffers containing sodium ions.
  • Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent.
  • Methylcellulose is preferred because it is readily and economically available and is easy to work with.
  • suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount which will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.
  • Compositions within the scope of this invention can contain a humectant to inhibit drying of the mucous membrane and to prevent irritation.
  • humectants any of a variety of pharmaceutically acceptable humectants can be employed including, for example sorbitol, propylene glycol or glycerol.
  • concentration will vary with the selected agent, although the presence or absence of these agents, or their concentration, is not an essential feature of the invention.
  • Enhanced absorption across the mucosal and especially nasal membrane can be accomplished employing a pharmaceutically acceptable surfactant.
  • useful surfactants for compositions include polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides such as Tween 80, Polyoxyl 40 Stearate, Polyoxyethylene 50 Stearate and Octoxynol. The usual concentration is form 1% to 10% based on the total weight.
  • a pharmaceutically acceptable preservative can be employed to increase the shelf- life of the compositions.
  • Benzyl alcohol may be suitable, although a variety of preservatives including, for example, Parabens, thimerosal, chlorobutanol, or bezalkonium chloride may also be employed.
  • a suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.
  • compositions must be selected to be chemically inert with respect to the lipooliogosaccharide. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure.
  • compositions of this invention are prepared by mixing the ingredients following generally accepted procedures.
  • the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
  • the pH may be from about 3 to 7.5.
  • Compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the mucosal route of administration. Dosages for humans or other mammals can be determined without undue experimentation by the skilled artisan from experiments involving mice, rabbits, chinchillas, etc.
  • the vaccine composition which is administered intranasally as provided herein may be formulated in any convenient manner and in a dosage formulation consistent with the mode of administration and the elicitation of a protective response.
  • the quantity of antigen to be administered depends on the subject to be immunized and the form of the antigen. Precise amounts and form of the antigen to be administered depend on the judgement of the practitioner. However, suitable dosage ranges are readily determinable by those skilled in the art and may be of the order of micrograms to milligrams. Suitable regimes for initial administration and booster doses also are variable, but may include an initial administration followed by subsequent administrations.
  • the lipooligosaccharides may conventionally be used in the preparation of the medicament e.g., vaccine.
  • the lipooligosaccharides may be formulated with a diluent or a pharmaceutically acceptable carrier e.g., a buffer or a saline.
  • the vaccine may additionally contain usual ingredients such as a stabilizer or as already mentioned above, a mucosal adjuvant or delivery system.
  • these products are selected according to standard pharmaceutical practices as described in Remington 's Pharmaceutical Sciences, a reference book in the field.
  • the vaccine may be administered by the mucosal route, as a unique dose or preferably, several times e.g., twice, three or four times at week or month intervals, according to a prime/boost mode.
  • the appropriate dosage depends upon various parameters, including the number of valencies contained in the vaccine, the serotypes of the lipooligosaccharides and the age of the recipient. It is indicated that a vaccine dose suitably contain per valency, from 0.5 to 100 ⁇ g, preferably from 1 to 50 ⁇ g, more preferably from 1 to 10 ⁇ g of lipooligosacchari.de.
  • a dose is advantageously under a volume of from 0.1 to 2 ml.
  • the vaccination protocol may be a strict mucosal protocol or a mix protocol in which the priming dose of the vaccine is administered by the mucosal e.g., intranasal route and the boosting dose(s) is (are) parenterally administered or vice versa.
  • Intranasal Immunization with Lipooligosaccharide-based Conjugate Vaccine from Nontypeable Haemophilus influenzae Inhibits Bacterial Colonization in Mouse
  • intranasal immunization with such a detoxified LOS-tetanus toxoid (dLOS-TT) vaccine would generate protective immunity against NTHi in a mouse model of nasopharyngeal colonization.
  • dLOS-TT detoxified LOS-tetanus toxoid
  • CT adjuvant cholera toxin
  • LOS-specific IgA antibody forming cells were also found in mucosal and lymphoid tissues with the highest number in nasal passage (528 per 10 6 cells).
  • the intranasal immunization elicited a significant rise of LOS-specific IgG (32-fold) and IgA (13-fold) in serum.
  • the vaccine group showed a significant reduction of NTHi by 74% and 76%, compared to that of control groups with CT alone or dLOS plus CT (p ⁇ 0.05). Negative correlations were found between bacterial counts and the levels of nasal wash IgA or IgG, saliva IgA or serum IgG.
  • NTHi LOS and conjugate vaccine Female BALB/c mice (6 weeks) were purchased from Taconic farms Inc. (Germantown, NY). The mice were in an animal facility in accordance with National Institutes of Health guidelines under animal study protocol 1009-01.
  • NTHi LOS and conjugate vaccine NTHi strain 9274 and five prototype strains 1479, 2019, 3198, 5657 and 7502 were obtained from M.A. Apicella, University of Iowa (Campagnari, A.A. et al. 1987 Infect Immun 55:882-887).
  • LOS of NTHi strain 9274 was extracted from cells by hot phenol water, and then purified by gel filtration as described previously (Gu, X.X. et al. 1995 Infect Immun 63:4115-4120).
  • Protein content was about 1% and nucleic acid content was less than 1 %.
  • Detoxification of the LOS, conjugation of dLOS to TT, and characterization of dLOS-TT from strain 9274 were described previously (Gu, X.X. et al. 1996 Infect Immun 64:4047-4053).
  • the composition of dLOS-TT was 638 ⁇ g of dLOS and 901 ⁇ g of TT per ml with a molar ratio of dLOS to TT at 35:1.
  • the strain was grown on chocolate agar at 37°C under 5% CO 2 for 8 h and transferred to 200 ml of 3% brain heart infusion medium (Difco Laboratories, Detroit, Mich.) containing NAD (5 ⁇ g/ml) and hemin (2 ⁇ g/ml) (Sigma Chemical Co., St. Louis, Mo.) in a 500-ml bottle.
  • the bottle was incubated at 150 ⁇ m in an incubator shaker (model G-25; New Brunswick Scientific, Co. Edison, N.J.) at 37°C overnight.
  • the culture was transferred to five 2.8-liter baffled Fernbach flasks, each of which contained 1.4 liters of the same medium.
  • LOS was purified from cells by a modified phenol-water extraction (Gu, X.X. et al. 1995 Infect Immun 63:4115-4120) and from the culture supernatant by gel filtration (Gu, X.X. and Tsai, CM. 1993 Anal Biochem 196:311-318).
  • the protein and nucleic acid contents of both purified LOSs were less than 1% (Smith, P.K. et al. 1985 Anal Biochem 150:76-85; Warburg, O. and W. Christian 1942 Biochem Z 310:385-421). Detoxification of LOS.
  • LPSs lipoprotein A
  • LOS 160 mg
  • each lot was dried over P 2 O 5 for 3 days, suspended in 16 ml of anhydrous hydrazine (Sigma), and incubated at 37°C for 2 h with mixing every 15 min. This suspension was cooled on ice and added dropwise to cold acetone in an ice bath until a precipitate formed (>90% acetone). The mixture was centrifuged at 5,000 x g at 5°C for 30 min.
  • the pellet was washed twice with cold acetone and dissolved in pyrogen-free water at a final concentration of 20 mg/ml.
  • the reaction mixture was ultracentrifuged at 150,000 x g at 5°C for 3 h.
  • the supernatant was freeze- dried and passed through a column (1.6 by 90 cm) of Sephadex G-50 (Pharmacia LKB Biotechnology, Uppsala, Sweden), eluted with 25 mM ammonium acetate, and monitored with a differential refractometer (R-400; Waters, Milford, Mass.).
  • the eluate was assayed for carbohydrate by the phenol-sulfuric acid method (Dubois, M. et al.
  • dLOS 70 mg was dissolved in 7 ml of 345 mM ADH (the molar ratio of ADH to LOS is -100:1 based on an estimated 3,000 M ⁇ for dLOS) (Gibson, B.W. et al. 1993 J Bacteriol 175:2702-2712; Helander, J.M. et al. 1988 Eur J Biochem 177:483- 492).
  • N-Hydroxysulfosuccinimide was added to a concentration of 8 mM, the pH was adjusted to 4.8 with 1 M HCl, and EDC was added to a concentration of 0.1 M.
  • the reaction mixture was stirred and maintained at pH 4.8 ⁇ 0.2 with 1 M HCl for 3 h at room temperature. It was adjusted to pH 7.0 with ⁇ aOH and passed through the G-50 column as described above.
  • the eluate was assayed for carbohydrate and for AH by a modification of a previously described method (Kemp, A.H. and M.R.A. Morgan 1986 J Immunol Methods 94:65-72) by measuring the A 490 of AH groups.
  • the peaks containing both carbohydrate and AH were pooled, freeze-dried three times to remove the salt, and designated AH-dLOS.
  • AH-dLOS was measured for its composition with dLOS and ADH as standards (Dubois, M. et al. 1956 Anal Biochem 28:250-256; Kemp, A.H. and M.R.A. Morgan 1986 J Immunol Methods 94:65-72).
  • TT was obtained from Connaught Laboratories, Inc., Swiftwater, Pa.
  • HMP was purified from ⁇ THi 12 (Barenkamp, S.J. 1996 Infect Immun 64:1246-1251).
  • AH-dLOS was coupled to carboxyl groups on TT or HMP at pH 5.6 with EDC.
  • AH-dLOS (20 mg) was dissolved in 2 ml of water and mixed with 10 mg of TT (5.9 mg/ml) or with 8 mg of HMP (4 mg/ml). The molar ratio of AH-dLOS to both TT (M ⁇ 150,000) and HMP (M ⁇ 120,000) was -100:1.
  • the pH was adjusted to 5.6 with 0.1 M HCl, and EDC was added to a concentration of 0.1 M.
  • the reaction mixture was stirred for 1 to 3 h at room temperature; the pH was maintained at 5.6 + 0.2 with 0.1 M HCl.
  • the reaction mixture was adjusted to pH 7.0, centrifuged at 1,000 x g for 10 min, and passed through a column (1.6 by 90 cm) of Sephacryl S-300 in 0.9% NaCl. Peaks that contained both protein and carbohydrate were pooled and designated dLOS-TT or dLOS- HMP. Both conjugates were analyzed for their composition of carbohydrate and protein with dLOS and bovine serum albumin (BSA) as standards (Dubois, M. et al. 1956 Anal Biochem 28:250-256; Smith, P.K. et al. 1985 Anal Biochem 150:76-85).
  • BSA bovine serum albumin
  • mice were immunized nasally with 10 ⁇ l of phosphate-buffered saline (PBS) containing a mixture of 5 ⁇ g of dLOS-TT and 1 ⁇ g of cholera toxin (List Biological Laboratories, Campbell, CA) as an adjuvant.
  • PBS phosphate-buffered saline
  • Control mice intranasally received 10 ⁇ l of PBS containing 5 ⁇ g of dLOS and/or 1 ⁇ g of CT.
  • Each dose was pipetted into the mouse nostril (5 ⁇ l each side) under anesthesia with intraperitoneal injection of 0.1 ml of 2% ketamine and 0.2 % xylazine. Immunizations were given 5 times on days 0, 7, 14, 21 and 28.
  • mice On day 35, one set of mice was used for bacterial challenge while another set was used for sample collections only described as follows. Nasal washes, saliva, bronchoalveolar lavage fluids (BALFs), fecal extracts, and sera were collected from mice of each group under anesthesia as described before (Kurono, Y. et al. 1999 J Infect Dis 180:122-132). Briefly, salivary samples were obtained following intraperitoneal injection with 0.1 ml of 0.1% piloca ⁇ ine (Sigma, St. Louis, MO) in PBS to induce salivary secretion. Blood samples were collected from axillary artery.
  • BALFs bronchoalveolar lavage fluids
  • fecal extracts fecal extracts
  • sera were collected from mice of each group under anesthesia as described before (Kurono, Y. et al. 1999 J Infect Dis 180:122-132). Briefly, salivary samples were obtained following intraperitoneal injection with 0.1 ml of 0.1% piloca ⁇
  • the nasal cavity was gently flushed from posterior opening of the nose with 200 ⁇ l of PBS and nasal washes were collected from the anterior openings of the nose.
  • BALF was obtained by irrigation with 1 ml of PBS through a blunted needle inserted into the trachea after incision.
  • Fecal extract samples were obtained by adding weighed pellets to PBS containing 0.01%o sodium azide (100 mg of fecal samples/ml) according to the method of deVos and Dick (Gu, X.X. et al. 1996 Infect Immun 64:4047-4053). Blood and fecal samples were centrifuged, and the supernatants were collected.
  • MNCs mononuclear cells
  • Detection of LOS-specific antibody-forming cells by enzyme-linked immunospot (ELISPOT) assay.
  • AFCs LOS-specific antibody-forming cells
  • ELISPOT enzyme-linked immunospot
  • 96-well filtration plates with a nitrocellulose base (Millititer HA; Millipore Co ⁇ ., Bedford, Mass.) were coated with 100 ⁇ l of strain 9274 LOS (10 ⁇ g/ml) and incubated overnight at 4°C The plates were washed three times with PBS and then blocked with complete medium for 1 h. After removing the blocking medium, test cells in complete medium were added at various concentrations and cultured at 37°C with 5% CO 2 for 6 h. After the incubation, the plates were washed thoroughly with PBS and then with PBS containing 0.05 % Tween 20 (PBS- T).
  • biotinylated goat anti-mouse IgA, IgG, or IgM was added in PBS-T at 1 : 1,000. After overnight incubation at 4°C, the plates were washed five times with PBS-T, and incubated with 5 ⁇ g/ml of avidin-peroxidase conjugates (Sigma) in PBS-T for 1 h at room temperature. After washing with PBS-T and PBS three times for each, spots were developed in 4-chloro-l-naphthol solution for 10 min. The reaction was stopped by washing with water. The plate were dried and dark blue-pu ⁇ le colored spots were counted as LOS-specific AFCs under a stereo microscope.
  • mice were euthanized on day 35 and then perfused transcardially with PBS, followed by perfusion with 10% neutral buffered formalin.
  • Mouse heads were removed and fixed in 10% fonnalin for 6 hr and decalcified with 0.12 M ethylenediamine tetraacetic acid (EDTA, pH 7.0) for 2 weeks. After dehydration, the tissues were embedded in paraffin.
  • EDTA ethylenediamine tetraacetic acid
  • Specimens were dehydrated through a graded series of ethanol and treated with 3% hydrogen peroxide in absolute methanol for 30 min. Sections were exposed to 5% normal goat serum in PBS for 30 min and then incubated overnight with biotinylated goat anti-mouse IgA, IgG, or IgM in 1% bovine serum albumin (BSA)-PBS. After rinsing with PBS, sections were incubated with avidin-biotin complex (Vector Laboratories, Burlingame, CA) for 1 h and developed in 0.05% 3,3'-diaminobenzidine-0.01% H 2 O 2 substrate medium in 0.1M PBS for 8 min.
  • BSA bovine serum albumin
  • mice immunized with different antigens were challenged with the homologous strain 9274.
  • the strain was grown on chocolate agar at 37°C under 5 % CO 2 for 16 h, and then 3 - 5 clones were transferred to another plate and incubated for 4 h.
  • a bacterial suspension was prepared to the concentration of 4 ⁇ 6 x 10 6 CFU/ml in PBS and stored on ice until use. The bacterial concentration was determined by a 65% transmission at wavelength 540 nm, and confirmed by counting the colonies after overnight incubation.
  • mice were intranasally inoculated with 10 ⁇ l of the bacterial suspension on day 35. Six hours postchallenge, nasal washes were collected and diluted serially in PBS, and 50 ⁇ l of the diluted samples were plated on chocolate agar. Bacterial colonies were counted after overnight incubation. To investigate correlation between antibody levels and bacterial clearance of strain 9274, saliva, BALF, fecal extract and serum samples were collected from each mouse simultaneously. To examine the effect of the vaccine on heterologous NTHi, strains 1479, 2019, 3198, 5657 and 7502 were used based on the same procedure except only one control group (CT) was included since no significant difference was found between control groups.
  • CT control group
  • the membranes were incubated with nasal wash or serum sample (1:10) for 3 h, followed by biotinylated goat anti-mouse IgA or IgG for 2h. The membranes were washed with TBS-T, and incubated with avidin- peroxidase conjugate for 1 h. After washing with TBS, the membranes were developed with 4-chloro-l-naphthol solution. A duplicate gel was silver-stained after SDS-PAGE.
  • TBS BSA-Tris buffered saline
  • mice from each group were given an intranasal immunization on days 0, 7, 14, 21 and 28 with dLOS-TT+CT, dLOS+CT or CT al External and serum samples were collected at 1 week after the last immunization.
  • ⁇ LOS antibodies were measured by ELISA using strain 9274 LOS as a coating antigen. Symbols: dLOS-TT+CT group versus either dL CT or CT group: p O.Ol.
  • 0 BALF Bronchoalveolar lavage fluid.
  • LOS-specific immune responses were elicited significantly by intranasal immunization with dLOS-TT and CT but not controls (Table 1).
  • LOS-specific IgA titers in external secretions and in serum were increased by dLOS-TT and CT, especially in nasal wash (90- fold), BALF (26-fold), saliva (13-fold) and serum (13-fold), whereas slight increase of LOS-specific IgA in fecal extract was found (3-fold) when compared to that of CT controls.
  • LOS-specific antibody-forming cells in mucosal effector tissues.
  • Intranasal immunization with dLOS-TT and CT resulted in detection of LOS-specific IgA AFCs in all tissues tested, including distant organs such as intestine and spleen (Table 2).
  • the majority of LOS-specific IgA AFCs were located in nasal passage (528 per 10 6 cells), followed by a small amount in other tested tissues.
  • the dominant isotype of LOS-specific AFCs was IgA, followed by small numbers of IgG but not IgM.
  • LOS-specific IgG AFCs were only detected in nasal passage, CLN and spleen.
  • Intranasal immunization with dLOS and CT elicited 2 LOS-specific IgA AFCs in nasal passage but not in other tested tissues. No AFC was found in any tissues from mice immunized with CT.
  • Immunohistochemical staining of the nose revealed that the mouse immunized with the dLOS-TT vaccine showed positive staining in the mucous blanket and glandular tissues (B) as compared with the control mouse (A). A large number of IgA-positive cells were found in nasal subepithehal layer and nasal glands. In contrast, staining with anti-IgG in the mouse immunized with the dLOS-TT vaccine was only seen in the area of the vessels but not the glandular tissue (C).
  • mice immunized with dLOS-TT and CT showed a significant reduction of bacterial recovery by 74% or 76% when compared to those of the mice immunized with CT alone or dLOS and CT (p ⁇ 0.05).
  • Relationship between LOS-specific antibody titers and bacterial counts from nasopharynx was further analyzed in nasal wash, saliva, BALF, fecal extract and serum from dLOS-TT and CT immunized and CT immunized mice.
  • Moraxella catarrhalis is a significant cause of otitis media in children.
  • Lipooligosaccharide (LOS) is a major surface antigen of M. catarrhalis and a potential vaccine candidate.
  • LOS Lipooligosaccharide
  • BALB/c mice were immunized intranasally with a mixture of dLOS -CRM (the diphtheria toxin cross- reactive mutant protein) and cholera toxin (CT) as an adjuvant, dLOS plus CT, or CT only. After immunization, the animals were aerosolly challenged with M. catarrhalis strain 25238.
  • dLOS-CRM Immunization with dLOS-CRM generated a significant increase in secreting IgA and IgG in nasal washes, lung lavage and saliva, and serum IgG, IgM and IgA against LOS of M. catarrhalis as detected by an indirect enzyme-linked immunosorbent assay (ELISA).
  • ELISA indirect enzyme-linked immunosorbent assay
  • the dLOS-CRM also elicited LOS-specific IgA, IgG, and IgM antibody-forming cells (AFCs) in different lymphoid tissues as measured by an enzyme-linked immunospot (ELISPOT) assay.
  • LOS-specific IgA AFCs were found in the nasal passages, spleens, nasal-associated lymphoid tissues (NALT), cervical lymph nodes (CLN), lungs, and small intestines.
  • LOS-specific IgG and IgM AFCs were only detected in the spleens, CLN and nasal passages.
  • the dLOS-CRM vaccine generated a significant bacterial clearance in the nasopharynx and lungs when compared to the controls (P ⁇ 0.01) following an aerosol challenge with the homologous strain 25238.
  • a comparison of dLOS-CRM, dLOS-TT and dLOS-UspA through intranasal immunization resulted in similar protection against M. catarrhalis.
  • intranasal immunization with dLOS-CRM containing CT showed a higher level of bacterial clearance in both sites when compared to subcutaneous injections with dLOS-CRM plus CT adjuvant.
  • dLOS-CRM induces specific mucosal and systemic immunity against M. catarrhalis through intranasal immunization, and provides effective bacterial clearance in the mouse nasopharynx and lungs. Therefore, it is envisioned as being an efficient route for vaccines to prevent otitis media and lower respiratory tract infections caused by M. catarrhalis.
  • mice Female BALB/c mice (6-8 weeks old) were purchased from Taconic farms Inc. (Germantown, NY).
  • Conjugate vaccine Purification of LOS from M. catarrhalis strain 25238, detoxification of the LOS, and conjugation of dLOS to carrier protein including CRM, TT, UspA were performed as described previously (Gu, X.X. et al. 1998 Infect Immun 66:1891- 1897).
  • Type A strain ATCC 25238 was grown on chocolate agar at 37°C in 5% CO 2 for 8 h and transferred to 250 ml of 3% tryptic soy broth (Difco Laboratories, Detroit, Mich.) in a 500-ml bottle. The bottle was incubated at 110 m in an incubator shaker (model G-25; New Brunswick Scientific Co., Edison, N.J.) at 37°C overnight. The culture was transferred to six 2.8-liter baffled Fernbach flasks, each of which contained 1.4 liters of tryptic soy broth. The flasks were shaken at 110 ⁇ m and maintained at 37°C for 24 h.
  • the culture was centrifuged at 15,000 x g and 4°C for 10 min to collect the cells.
  • the cell pellets were washed once with 95% ethanol, twice with acetone, and twice with petroleum ether (Masoud, H. et al. 1994 Can J Chem 72:1466- 1477) and dried to a powder.
  • the LOS was extracted from cells (Gu, X.X. et al. 1995 Infect Immun 63:4115-4120), and the protein and nucleic acid contents of the LOS were less than 1% (Smith, P. K. et al. 1985 Anal Biochem 150:76-85; Warburg, O., and W. Christian. 1942 Biochem Z 310:384-421). Detoxification of LOS.
  • Anhydrous hydrazine treatment of LOS removes esterified fatty acids from lipid A (Gu, X.X. et al. 1996 Infect Immun 64:4047-4053; Gupta, R. K. et al. 1992 Infect Immun 60:3201-3208).
  • LOS 160 mg was suspended in 16 ml of anhydrous hydrazine (Sigma Chemical Co., St. Louis, Mo.) and incubated at 37°C for 3 h with mixing. This suspension was cooled on ice and added dropwise with cold acetone until a precipitate formed. The mixture was centrifuged at 5,000 x g and 5°C for 30 min.
  • the pellet was washed twice with cold acetone, dissolved in pyrogen-free water at a final concentration of 10 to 20 mg/ml, and then ultracentrifuged at 150,000 x g and 5°C for 3 h.
  • the supernatant was passed through a column (1.6 by 90 cm) of Sephadex G-50 (Pharmacia LKB Biotechnology, Uppsala, Sweden) eluted with 25 mM ammonium acetate and monitored with a differential refractometer (R-400; Waters, Milford, Mass.).
  • the eluate was assayed for carbohydrate by a phenol-sulfuric acid method (Dubois, M. et al. 1956 Anal Biochem 28:250-256).
  • the carbohydrate-containing fractions were pooled, freeze- dried, and designated dLOS.
  • Adipic acid dihydrazide (ADH; Aldrich Chemical Co., Milwaukee, Wis.) was bound to dLOS to form adipic hydrazide (AH)-dLOS derivatives, using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) and N- hydroxysulfosuccinimide (sulfo-NHS) (Pierce) (Gu, X.X., and CM. Tsai 1993 Infect Immun 61:1873-1880).
  • ADH Adipic acid dihydrazide
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl
  • sulfo-NHS N- hydroxysulfosuccinimide
  • dLOS 70 mg was dissolved in 7 ml of 345 mM ADH (molar ratio of ADH to LOS is -100 to 1, based on an estimated M, of 3,000 for dLOS) (Edebrink, P. 1994 Carbohydr Res 257:269-284).
  • Sulfo-NHS was added to a concentration of 8 mM, the pH was adjusted to 4.8, and EDC was added to a concentration of 0.1 M.
  • the reaction mixture was stirred and maintained at pH 4.8 for 3 h.
  • the reaction mixture was adjusted to pH 7.0 and passed through the G-50 column as described above.
  • the eluate was assayed for carbohydrate and for AH (Kemp, A. H., and M. R. A.
  • AH-dLOS Conjugation of AH-dLOS to proteins.
  • TT was obtained from Connaught Laboratories Inc., Swiftwater, Pa., and HMP was purified from NTHi strain 12 (Barenkamp, S. J. 1996 Infect Immun 64:1246-1251).
  • AH-dLOS was coupled to TT or HMP to form conjugates (Gu, X.X., and CM. Tsai 1993 Infect Immun 61:1873-1880). Briefly, AH-dLOS (30 mg) was dissolved with 3 ml of water and mixed with 15 mg of TT (5.9 mg/ml) or with 12 mg of HMP (4 mg/ml).
  • the molar ratio of AH-dLOS to both TT (M r , 150,000) and HMP (M r , 120,000) was -100 to 1.
  • the pH was adjusted to 5.4, and EDC was added to a concentration of 0.05 to 0.1 M.
  • the reaction mixture was stirred, and the pH was maintained at 5.4 for 3 h.
  • the reaction mixture was adjusted to pH 7.0, centrifuged, and passed through a column (1.6 by 90 cm) of Sephacryl S-300 in 0.9% NaCl. Peaks that contained both protein and carbohydrate were pooled and designated dLOS-TT or dLOS-HMP.
  • mice 6-8 for each group, were immunized intranasally (i.n.) 4 times, or subcutaneously (s.c.) 3 times, with PBS or 5 ⁇ g of dLOS- protein at l ⁇ 2-week intervals, respectively.
  • the total volume of administration is 10 ⁇ l for i.n. inoculation, or 0.2 ml for s.c. injection with or without Ribi 700 (25 ⁇ g/mouse) or cholera toxin (CT, 1 ⁇ g/mouse) adjuvant.
  • Ribi 700 25 ⁇ g/mouse
  • CT cholera toxin
  • LOS-specific antibodies by ELISA.
  • Detection of LOS-specific antibody-forming cells AFCs). Mononuclear cells were taken from the nasal passage, spleen, nasal-associated lymphoid tissue, cervical lymph node, Peyer's patch and lung. Numbers of LOS-specific IgA-, IgG-, and IgM-producing cells in each tissue were determined by an enzyme-linked immunospot (ELISPOT) assay.
  • ELISPOT enzyme-linked immunospot
  • Bacterial aerosol challenge The bacterial aerosol challenges were carried out one week after the last immunization in an inhalation exposure system (Glas-col, Terre Haute, Ind.) (Hu, W.G. et al. 1999 Vaccine 18:799-804). Conditions were as follows: challenge dose of bacteria, 10 8 to 5xl0 8 CFU/ml in the nebulizer; nebulizing time, 40 min; vacuum flowmeter, 60 standard ft 3 /h; and compressed air flowmeter, 10 ft 3 /h.
  • mice lungs were removed, and homogenated in 5 ml of PBS for 1 min at low speed in a tissue homogenizer (Stomacher Lab System Model 80, Seward, London, UK).
  • tissue homogenizer Stomacher Lab System Model 80, Seward, London, UK.
  • nasal washes were obtained by flushing the nasal cavity with 200 ⁇ l of PBS.
  • the appropriately diluted or undiluted lung homogenates, and nasal washes were plated on chocolate agar plates, and the bacterial colonies were counted after overnight incubation, hi addition, sera, nasal washes and lung homogenates were collected for antibody quantification.
  • the viable bacteria were expressed as the geometric mean CFU of n independent observations + standard deviation. Geometric means of reciprocal antibody titers were determined. Significance was determined by Student's t test.
  • mice were intranasally immunized 4 times at 1-week intervals with 10 ⁇ l of PBS containing a mixture of 5 ⁇ g of dLOS-CRM and 1 ⁇ g of or 10 ⁇ l of PBS containing a mixture of 5 ⁇ g of dLOS and 1 ⁇ of CT, or 10 ⁇ l of PBS containing 1 ⁇ g of CT, respectively.
  • mice were challenged with 2 X 10 8 CFU of M. catarrhalis strain 25238 pe in a nebulizer, and nasal washes and lungs were collected at 6 h postchallenge, respectively.
  • mice were intranasally immunized 4 times at 1-week intervals with 10 ⁇ l of PBS containing a mixture of 5 ⁇ g of dLOS-CRM, dLOS-T dLOS-UspA and 1 ⁇ g of CT, or 10 ⁇ l of PBS containing 1 ⁇ g of CT, respectively.
  • mice were intranasally admimstered 4 times at 1-week intervals with 10 ⁇ l of PBS containing a mixture of 5 ⁇ g of dLOS-CRM and 1 ⁇ CT, or 10 ⁇ l of PBS containing 1 ⁇ g of CT, or subcutaneously injected 3 times at 2-week intervals with 0.2 ml of a mixture of 5 ⁇ g of dL CRM and 1 ⁇ g of CT, or 0.2 ml of PBS containing 1 ⁇ g of CT, respectively.
  • b Geometric mean (+ SD range) of eight mice.

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Abstract

La présente invention concerne une immunisation intranasale qui fait appel à un lipooligosaccharide détoxifié provenant d'un Haemophilus influenzae ou d'un Moraxella catarrhalis non typable.
PCT/US2001/032331 1996-04-23 2001-10-16 Immunisation intranasale par un lipooligosaccharide detoxifie provenant d'un haemophilus influenzae ou d'un moraxella catarrhalis non typable WO2002089839A1 (fr)

Priority Applications (3)

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CA002446355A CA2446355A1 (fr) 2001-05-03 2001-10-16 Immunisation intranasale par un lipooligosaccharide detoxifie provenant d'un haemophilus influenzae ou d'un moraxella catarrhalis non typable
US10/688,115 US20040126381A1 (en) 1996-04-23 2003-10-17 Intranasal immunization with detoxified lipooligosaccharide from nontypeable haemophilus influenzae or moraxella
US11/260,773 US7641906B2 (en) 1996-04-23 2005-10-27 Intranasal immunization with detoxified lipooligosaccharide from nontypeable Haemophilus influenzae or Moraxella catarrhalis

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US28869501P 2001-05-03 2001-05-03
US60/288,695 2001-05-03

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US09/610,034 Continuation-In-Part US6685949B1 (en) 1996-04-23 2000-07-05 Lipooligosaccharide based vaccine for prevention of moraxella (branhamella)catarrhalis infections in humans
US09/789,017 Continuation-In-Part US6607725B2 (en) 1996-04-23 2001-02-20 Conjugate vaccine for nontypeable Haemophilus influenzae

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US8551501B2 (en) 2001-08-16 2013-10-08 Mucovax Inc. Chitin microparticles and their medical uses

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