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WO1999036086A1 - Vaccin a base de lipooligosaccharide destine a la prevention d'infections dues a moraxella (branhamella) catarrhalis chez les mammaliens - Google Patents

Vaccin a base de lipooligosaccharide destine a la prevention d'infections dues a moraxella (branhamella) catarrhalis chez les mammaliens Download PDF

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Publication number
WO1999036086A1
WO1999036086A1 PCT/US1999/000590 US9900590W WO9936086A1 WO 1999036086 A1 WO1999036086 A1 WO 1999036086A1 US 9900590 W US9900590 W US 9900590W WO 9936086 A1 WO9936086 A1 WO 9936086A1
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WIPO (PCT)
Prior art keywords
dlos
los
catarrhalis
hmp
isolated
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PCT/US1999/000590
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English (en)
Inventor
Xin-Xing Gu
John B. Robbins
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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 AU22212/99A priority Critical patent/AU2221299A/en
Application filed by The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services filed Critical The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority to BR9906902-4A priority patent/BR9906902A/pt
Priority to IL13690399A priority patent/IL136903A0/xx
Priority to JP2000539859A priority patent/JP2002509115A/ja
Priority to KR1020007007737A priority patent/KR20010034124A/ko
Priority to CA002315746A priority patent/CA2315746A1/fr
Priority to EP99902170A priority patent/EP1047447A1/fr
Publication of WO1999036086A1 publication Critical patent/WO1999036086A1/fr
Priority to US09/610,034 priority patent/US6685949B1/en
Priority to AU2003200964A priority patent/AU2003200964B2/en
Priority to US10/688,115 priority patent/US20040126381A1/en
Priority to US10/729,027 priority patent/US20040115214A1/en
Priority to US11/260,773 priority patent/US7641906B2/en

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Classifications

    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • A61K2039/5555—Muramyl dipeptides
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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 present invention relates to conjugate vaccines for prevention of bacterial infections. More specifically, the invention relates to a conjugate vaccine for Moraxella (Branhamella) catarrhalis infections in humans, comprising lipooligosaccharide derived from the bacteria, from which esterified fatty acids or lipid A has been removed, which is linked to an immunogenic carrier.
  • a conjugate vaccine for Moraxella (Branhamella) catarrhalis infections in humans comprising lipooligosaccharide derived from the bacteria, from which esterified fatty acids or lipid A has been removed, which is linked to an immunogenic carrier.
  • Moraxella (Branhamella) catarrhalis is a pathogenic bacterium, recognized as the third most common causative agent of otitis media and sinusitis in children, after Streptococcus pneumoniae and Haemophilus influenzae (Bluestone, CD., 1986, Drugs 31(Suppl 3):132-41; Catlin, B.W., 1990, Clin. Microbiol. Rev. 3:293-320; Doern, G.N., 1986, Diagn. Microbiol. Infect. Dis. 4:191-201; Enright, M.C. & H. McKenzie, 1997, J.
  • M. catarrhalis The incidence of disease caused by M. catarrhalis appears to be increasing (McLeod, D.T., et al., 1986, Br. Med. J. 292:1103-1105; Fung, C.P., et al., 1992, J. Antimicrob. Chemother. 30:47-55).
  • M. catarrhalis-mediated diseases There is no vaccine for M. catarrhalis- mediated diseases.
  • protective antigens of M. catarrhalis have not been clearly defined, development of serum antibodies against M. catarrhalis appears to be important in immunity against M. catarrhalis.
  • M. catarrhalis Antibodies to M. catarrhalis have also been detected in acute and in convalescent sera of adult patients (Christensen, J.J., et al., 1990, Clin. Diagn. Lab. Immunol. 3:717-721; Rahman, M., et al., 1997, APMIS 105:213-220). Most convalescent sera demonstrate bactericidal activity against the corresponding M. catarrhalis isolate (Chapman, A.J.Jr., et al., 1985,
  • OMP outer membrane proteins
  • CD protein has been cloned and sequenced (Murphy et al., 1993, Molec. Microbiol. 10(1):87).
  • Other outer membrane proteins that have been purified and characterized include protein E (OMP E) (Bhushan et al., 1994, J. Bacteriol., 176(21):6636), protein Bl (Ducey et al., 1996, Abstracts, Gen. Mtg. Am. Soc. Microbiol., 96(0): 186), and protein COPB (Aebi et al., 1996, Abstracts, Intersci. Conf. Antimicrobial Agents & Chemotherapy 36:158).
  • OMP E protein E
  • protein Bl Ducey et al., 1996, Abstracts, Gen. Mtg. Am. Soc. Microbiol., 96(0): 186
  • protein COPB Aebi et al., 1996, Abstracts, Intersci. Conf. Antimicrobial Agents & Chem
  • Lipooligosaccharide a major surface component of M. catarrhalis, is a virulence factor for the pathogenesis of the bacterial infections (Doyle, W.J., 1989,
  • the LOS may be important for development of immunoprotection because (1) serum antibodies to LOS have been detected in patients with M. catarrhalis infections, (2) the convalescent-phase IgG anti-LOS from patients has demonstrated bactericidal activity against M. catarrhalis strains, and (3) LOS appears to have a conserved structure based on its serological properties in humans (Rahman, M., et al., 1995, Eur. J. Clin. Microbiol. Infect. Dis.
  • LOS Three major antigenic types (A, B and C) of LOS account for about 95% of M. catarrhalis strains (i.e., 61% A; 29% B; and 5% C in one study) (Naneechoutte, M., et al., 1990, J. Clin. Microbiol. 28:182-187). Studies have shown that these LOSs contain an oligosaccharide linked to lipid A, without an O-specific polysaccharide, and the oligosaccharides from the three serotypes are branched with a common inner core (Edebrink, P., et al., 1994, Carbohydr. Res.
  • Lipopolysaccharide (LPS) and LOS from a variety of microorganisms are generally toxic in vivo to mammals. Many approaches have been used to detoxify LPS or LOS, or to obtain nontoxic polysaccharides from LPS or oligosaccharides from LOS.
  • Conjugate vaccines made up of a carbohydrate-containing antigen bound to an immunomodulating cytokine, lymphokine, hormone or growth factor have been disclosed in U.S. Pat. No. 5,334,379.
  • Canadian Pat. No. 2,162,193 discloses that a purified bacterial lactoferrin receptor protein may be used as a vaccine against pathogens that produce a lactoferrin receptor protein, including M. catarrhalis.
  • PCT Application WO 90/11777 discloses a method for obtaining unassembled bacterial pilus subunits for use in a vaccine against M. catarrhalis and other bacteria.
  • a vaccine against M.catarrhalis that is both nontoxic and immunogenic is needed to prevent otitis media, sinusitis and similar respiratory tract infections in mammals, particularly in human children and adults.
  • the detoxified products i.e., hapten
  • the detoxified products are generally poorly immunogenic in vivo. Therefore, there is a need for a form of M. catarrhalis LOS that is detoxified but sufficiently immunogenic to elicit an immune response with production of anti-LOS antibodies, preferably IgG, in vivo in mammals.
  • conjugate vaccine for Moraxella catarrhalis including a lipooligosaccharide (LOS) isolated from M. catarrhalis and detoxified by treating to remove esterified fatty acids to produce a detoxified LOS (dLOS), or by treating to remove lipid A to produce an oligosaccharide (OS), and an immunogenic carrier covalently linked thereto.
  • the immunogenic carrier is a protein.
  • the immunogenic carrier protein is selected from the group consisting of UspA isolated from M. catarrhalis, CD isolated from M.
  • catarrhalis tetanus toxin/toxoid
  • HMP high molecular weight protein isolated from nontypeable Haemophilus influenzae, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus NP 7 protein; CRMs (Cross Reacting Materials), including CRM 197 (Pappenheimer et al., Immunochem.
  • the immunogenic carrier protein is tetanus toxoid or HMP.
  • a pharmaceutical composition that includes such a vaccine conjugate in a pharmaceutically acceptable carrier, which may include an adjuvant.
  • the adjuvant is an admixture of monophosphoryl lipid A and trehalose dimycolate or alum.
  • the immunogenic carrier is covalently linked to de-esterified LOS via a linker compound.
  • the linker compound is selected from the group consisting of adipic acid dihydrazide, ⁇ -aminohexanoic acid, chlorohexanol dimethyl acetal, D- glucuronolactone and p-nitrophenylethyl amine, and more preferably, the linker compound is adipic acid dihydrazide.
  • the vaccine further includes an oligosaccharide (OS) isolated from M. catarrhalis by removal of lipid A from LOS, which is covalently linked to an immunogenic carrier.
  • OS oligosaccharide
  • the Moraxella catarrhalis from which the lipooligosaccharide is isolated is a purified strain of Moraxella catarrhalis.
  • a method of preventing otitis media caused by infection with Moraxella catarrhalis in a mammal including administering to the mammal an effective immunoprotective amount of the conjugate vaccine that includes a detoxified lipooligosaccharide (dLOS) produced by de- esterification of LOS derived from Moraxella catarrhalis, or an oligosaccharide (OS) produced by removal of lipid A from LOS, and an immunogenic carrier covalently linked to the dLOS or to the OS.
  • dLOS detoxified lipooligosaccharide
  • OS oligosaccharide
  • the mammal is a human.
  • the conjugate vaccine is administered parenterally.
  • the conjugate vaccine is administered by intramuscular injection, subcutaneous injection, or by deposit on intranasal mucosal membrane or combinations thereof.
  • the effective immunoprotective amount is between about 10 ⁇ g and about 50 ⁇ g per dose.
  • the method may also include injecting between about 10 ⁇ g and about 25 ⁇ g of the conjugate vaccine at about two months and again at about thirteen months after the administering step.
  • the administering step includes administering a first dose, and then administering a second dose of about 10 ⁇ g to about 25 ⁇ g of the conjugate vaccine at about two months after the first dose, administering a third dose of about 10 ⁇ g to about 25 ⁇ g of the conjugate vaccine at about 2 months after the second dose, and administering a fourth dose of about 10 ⁇ g to about 25 ⁇ g of the conjugate vaccine at about 12 months after the third dose.
  • LOS lipooligosaccharide isolated from Moraxella catarrhalis, including removing ester-linked fatty acids from the LOS.
  • the ester-linked fatty acids are removed with hydrazine or a mild alkaline reagent.
  • the invention also includes a method for detoxifying LOS from Moraxella catarrhalis, including removal of lipid A from the LOS to produce OS.
  • the lipid A is removed by acid treatment.
  • a method of making a conjugate vaccine against Moraxella catarrhalis including removing ester- linked fatty acids from lipooligosaccharide (LOS) isolated from M. catarrhalis to produce de-esterified LOS (dLOS); and covalently linking the dLOS to an immunogenic carrier.
  • LOS lipooligosaccharide
  • dLOS de-esterified LOS
  • the removing step comprises treating the LOS with hydrazine or a mild alkaline reagent.
  • the linking step includes attaching the dLOS to a linker compound and attaching the linker compound to the immunogenic carrier.
  • the linker compound is adipic acid dihydrazide, ⁇ -aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone or p-nitrophenylethyl amine, and more preferably, the linker compound is adipic acid dihydrazide.
  • the vaccine composition may include an adjuvant.
  • the present invention also provides a conjugate vaccine comprising a lipooligosaccharide (LOS) isolated from M. catarrhalis and detoxified by treating to remove esterified fatty acids to produce detoxified LOS (dLOS), or by removing lipid A to produce oligosaccharide (OS), and an immunogenic carrier covalently linked thereto, for use in preventing otitis media caused by infection with Moraxella catarrhalis in a mammal.
  • the immunogenic carrier is a protein.
  • immunogenic carrier protein is UspA isolated from M. catarrhalis, CD isolated from M.
  • tetanus toxin/toxoid a high molecular weight protein isolated from nontypeable Haemophilus influenzae, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus NP 7 protein; CRMs including CRM 197 (Pappenheimer et al. supra.) and CRM 3201 (Black et al., supra.); or respiratory syncytial virus F and G protein.
  • the immunogenic carrier protein is tetanus toxoid or HMP.
  • FIG. 1 graphically shows the bactericidal titers against M. catarrhalis strain 25238 of rabbit antisera obtained from groups of two to three rabbits, in which each member of the group was individually vaccinated twice with: LOS ("LOS"), conjugates
  • dLOS-TT and "dLOS-HMP"
  • dLOS-TT + Ribi or conjugates with adjuvant
  • dLOS-TT + Ribi or conjugates with adjuvant
  • dLOS-TT + Ribi or conjugates with adjuvant
  • dLOS-TT + Ribi or conjugates with adjuvant
  • dLOS-TT + Ribi or conjugates with adjuvant
  • dLOS-TT + Ribi or conjugates with adjuvant
  • dLOS-TT + Ribi and "dLOS -HMP + Ribi”
  • the bactericidal titers are shown as the fold increase above the value for preimmune sera, based on the serum dilution causing more than 50% killing of the bacteria and expressed as the geometric mean (the bar) and standard deviation (the line above the bar) for each group.
  • the bactericidal titre of hyperimmune sera elicited by M. catarrhalis whole cells was 1:1,600.
  • FIG. 2 is a schematic diagram of a passive protection study in a mouse pulmonary clearance model using aerosol challenge of M. catarrhalis strain 25238. Forty mice were immunized with either rabbit antisera against dLOS-TT, or with pre- immune sera, then challenged with M. catarrhalis by aerosol chamber 18 hours after immunization. The mice were sacrificed at 3 and 6 hours after challenge.
  • FIG. 3 is a graph showing the results of the passive protection study described in the legend to FIG. 2. Lungs and blood samples were collected for analysis. The y-axis shows the bacterial colony forming units (CFU) per lung. The first bar shows the control group, and the second group shows the vaccine group. At three hours post- challenge, the amount of bacteria in the vaccine group was reduced by 50% compared to the control. At 6 hours post-challenge, there was a 61% reduction in the vaccine group compared to the control group.
  • CFU colony forming units
  • Lipooligosaccharide (LOS) of Moraxella (Branhamella) catarrhalis is a major surface antigen that elicits bactericidal antibodies against bacteria that cause otitis media and sinusitis in children and respiratory tract infections in adults.
  • the bacteria are referred to hereinafter as Moraxella catarrhalis or M. catarrhalis.
  • the M. catarrhalis LOS was isolated and treated to reduce its toxicity by about 20,000-fold, as assayed using a Limulus amebocyte lysate (LAL) test.
  • LAL Limulus amebocyte lysate
  • the detoxified LOS (dLOS) was coupled to a carrier (e.g., tetanus toxoid or high-molecular-weight proteins purified from nontypeable Haemophilus influenzae) through a linker compound to form dLOS- TT or dLOS-HMP.
  • a carrier e.g., tetanus toxoid or high-molecular-weight proteins purified from nontypeable Haemophilus influenzae
  • the molar ratios of dLOS to TT and HMP in the resulting conjugates were about 19:1 and 31 :1, respectively.
  • the antigenicity of the two conjugates was similar to that of isolated LOS, as determined by a double- immunodiffusion assay.
  • IgG immunoglobulin G
  • mice a 50- to 100-fold rise in the mean IgG levels was detected after three injections of the conjugates, and in rabbits, a 350- to 700-fold rise of IgG levels was detected after two injections.
  • the immunogenicity of the conjugate was enhanced by inclusion of an adjuvant in the conjugate formulation.
  • catarrhalis strain 25238 was grown on chocolate agar for 8 hr, and then inoculated into 3% tryptic soy broth (TSB) which was incubated with shaking at 37°C overnight. The culture was further diluted and transferred to baffled flasks containing TSB, and grown with shaking at 37°C for an additional 24 hr. The cells were collected by centrifugation, and the pelleted cells were washed with ethanol, acetone, and petroleum ether using standard methods (as described in Masoud, H., et al., 1994, Can. J. Chem. 72:1466-1477), before being dried to a powder.
  • the LOS was extracted from cells by a standard hot phenol-water method (Westphal, O., et al., 1965, Methods Carbohydr. Chem. 5:83-91) with modifications (Gu, X-X., 1995, Infect. Immun. 63:4115-4120) to yield LOS with a protein and nucleic acid content of less than 1%
  • hydrazine for detoxification of LOS from M. catarrhalis
  • any reagent or enzyme capable of removing esterified fatty acids from lipid A such as mild alkaline treatment, i.e., treatment with dilute (0.1 N) NaOH or other dilute aqueous base solutions having a pH of between about 13.2 and 13.6, is within the scope of the present invention. It is important that the detoxification conditions be mild enough to not hydrolyze the oligosaccharide portion of the LOS. Hydrolysis of the oligosaccharide will destroy protective epitopes.
  • the isolated M is important that the detoxification conditions be mild enough to not hydrolyze the oligosaccharide portion of the LOS. Hydrolysis of the oligosaccharide will destroy protective epitopes.
  • LOS was detoxified using anhydrous hydrazine treatment under mild conditions substantially as previously described (Gu, X.X., et al., 1996, Infect. Immun. 64:4047-4053; Gupta, R.K., et al., 1992, Infect. Immun. 60:3201-3208). Briefly, LOS was suspended in anhydrous hydrazine and incubated at a temperature of between 1°C and 100°C, preferably between 25°C and 75°C, and more preferably, about 37°C
  • Incubation with mixing was between 10 min to 24 hr, preferably about 2 hr to about 3 hr, and then the mixture was cooled and cold acetone was added until a precipitate formed which was collected by centrifugation.
  • the pellet was washed with acetone, dissolved in water, and then ultracentrifuged.
  • the supernatant obtained after ultracentrifugation was freeze-dried, redissolved and subjected to column chromatography to elute the carbohydrate-containing fractions, which were pooled, freeze-dried, and designated dLOS.
  • the dLOS was about 38 % of LOS.
  • LOS can be detoxified by mild acid treatment using dilute or weak aqueous acids having a pH of between about 2 and 3, as disclosed by Gu et al. (Infect. Immun. 61:1873-1880, 1993) which results in removal of lipid A to produce an oligosaccharide (OS).
  • This OS is then conjuated to carriers using the same methods as for dLOS.
  • acetic acid for removal of lipid A from M. catarrhalis LOS is described herein, the use of any reagent or enzyme capable of removing lipid A is within the scope of the present invention.
  • the OS-protein conjugates are also immunogenic in both mice and rabbits, and elicit antibodies to both LOS and the carrier proteins.
  • conjugate(with adjuvant)-immunized sera showed bactericidal activity against the homologous M. catarrhalis strain 25238.
  • conjugate-immunized sera showed bactericidal activity against homologous strain 25238.
  • the dLOS may be directly covalently bonded to a carrier protein, for example, by using a cross-linking reagent such as glutaraldehyde.
  • a cross-linking reagent such as glutaraldehyde.
  • the dLOS or OS conjugates are produced by use of a linker compound separating the dLOS or OS and the carrier, using any of a variety of known methods (e.g., see Marburg et al., 1986, J. Am. Chem. Soc. 108:5282, and U.S. Pat. Nos. 4,882,317; 5,153,312; 5,204,098). Presence of a linker promotes efficient coupling of the dLOS or OS to the carrier and optimizes immunogenicity of the conjugate.
  • Linkers having chains whose length and flexibility can be adjusted as desired may separate the carbohydrate and carrier components. Linkers may permit increased translational and rotational characteristics of the conjugate antigens, thus increasing access of the binding sites of antibodies. Between the bifunctional sites, the linker chains may contain a variety of structural features, including heteroatoms and cleavage sites. Although adipic dihydrazide (ADH) is a preferred linker, other suitable linkers include, for example, heterodifunctional linkers such as ⁇ -aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenyl amine.
  • ADH adipic dihydrazide
  • Coupling reagents contemplated for use in the present invention include hydroxysuccinimides and carbodiimides. Many suitable linkers and coupling reagents are known to those of ordinary skill in the art (Dick et al., Conjugate Vaccines, J. M. Cruse & R.E. Lewis, Jr., eds., Karger, New York, pp. 48-114, 1989).
  • the dLOS or OS was first derivatized with adipic dihydrazide (ADH) which serves as the linker to a protein carrier.
  • ADH adipic acid dihydrazide
  • ADH was bound to the carboxyl group of Kdo moiety of the dLOS or OS to form AH-dLOS or AH-OS derivatives using known methods (Gu, X.X., & CM. Tsai, 1993, Infect. Immun. 61:1873-1880).
  • a molar excess of ADH was used to ensure more efficient coupling and to limit dLOS-dLOS coupling.
  • the molar ratio of ADH to dLOS or OS is typically between about 10:1 and about 250:1, preferably between about 50:1 and about 150:1, and more preferably, about 100:1.
  • one ADH per dLOS or OS is present in the AH-dLOS conjugate.
  • the molar ratio of dLOS or OS to carrier is between about 15 and about 100, in a preferred lower range of about 20 to about 35 and a preferred upper range of about 40 to about 75, preferably between about 25 and about 50, and more preferably about 50. This ratio is generally controlled by varying the starting concentrations of AH-dLOS or AH-OS and carrier, and the time of reaction.
  • the derivatized dLOS or OS was purified from the reaction mixture by column chromatography to obtain eluate fractions containing both carbohydrate and adipic hydrazide (Kemp, A.H. & M.R.A. Morgan, 1986, J. Immunol. Methods 94:65-72). These fractions were pooled, freeze-dried, and designated AH- dLOS or AH-OS.
  • HMP high molecular weight proteins
  • the carrier increases the immunogenicity of the oligosaccharide and antibodies raised against the carrier may be medically beneficial.
  • the carrier may be water soluble or insoluble.
  • Suitable natural or synthetic polymeric immunogenic carriers include, for example, materials containing a primary and/or secondary amino group, an azido group or a carboxyl group.
  • immunogenic carrier proteins may be used to produce the dLOS- or OS-carrier conjugates of the present invention.
  • proteins include, for example, 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 (known as cross-reacting materials or CRMs) and other proteins.
  • Preferred outer membrane proteins are those isolated from gram-negative bacteria.
  • Preferred outer membrane proteins include UspA and CD isolated from M. catarrhalis outer membrane. Toxoids are also preferred.
  • Nonlimiting examples of bacterial toxins and toxoids contemplated for use in the present invention include, for example, tetanus toxin or toxoid, diphtheria toxin or toxoid, detoxified P. aeruginosa toxin A, cholera toxin or toxoid, pertussis toxin or toxoid, and Clostridium sp. exotoxin or toxoid.
  • viral proteins e.g., hepatitis B surface or core antigens; rotavirus NP 7 protein and respiratory syncytial virus (RSN) F and G proteins
  • CRMs include CRM 197 , antigenically equivalent to diphtheria toxin (Pappenheimer et al., supra.) and CRM3201, a genetically manipulated variant of pertussis toxin (Black et al., supra.).
  • immunogenic carrier proteins from non- mammalian sources, such as, for example, keyhole limpet hemocyanin, horseshoe crab hemocyanin and plant edestin is also within the scope of the invention.
  • dLOS-or OS-protein conjugates Many coupling methods are envisioned for producing the M. catarrhalis dLOS-or OS-protein conjugates.
  • dLOS or OS was derivatized with A ⁇ and then linked to TT or ⁇ MP.
  • another method for producing suitable dLOS- or OS-protein conjugates involves cystamine derivatization of dLOS, by EDC-mediated derivatization, followed by disulfide conjugation to ⁇ -succimidyl-3-(2- pyridyldithio) propionate-derivatized protein.
  • Other well-known methods for conjugating oligosaccharides to immunogenic carrier proteins are also within the scope of the invention, as described, for example, in U.S. Patent No. 5,153,312, U.S. Patent No. 5,204,098; and European Patents EP 0497 525; and EP 0 245 045.
  • AH-dLOS or AH-OS was coupled to tetanus toxoid (TT) or high molecular weight proteins (HMP) from H. influenzae to form conjugates (Gu, X.X., & CM. Tsai,
  • the molar ratio of AH-dLOS or AH-OS to the protein component in the reaction mixture is typically between about 10:1 and about 250:1, preferably is between about 50:1 and about 150:1, and more preferably, is about 100:1.
  • AH-dLOS dissolved in water, was mixed with TT or HMP at molar ratios of AH-dLOS to conjugating protein of about 100:1.
  • the pH was adjusted to 5.4 ⁇ 0.2 and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide HC1 was added to the stirred reaction mixture for 1 hr to 3 hr.
  • the reaction mixture was adjusted to pH 7.0, centrifuged, and purified by column chromatography. Peaks that contained both protein and carbohydrate were pooled, and designated as dLOS-TT or dLOS-HMP, depending on the protein used in the conjugate. Conjugates were analyzed for their carbohydrate and protein compositions using conventional methods, and dLOS and BSA as standards (Dubois, M., et al., 1956, Anal. Biochem. 28:250-256; Smith, P.K., et al, 1985, Anal. Biochem. 150:76-85).
  • the dLOS or OS coupled to the carrier may have originated with a single M. catarrhalis strain or with a variety of strains, to produce a multivalent mixture.
  • dLOS- or OS-carrier conjugates may be prepared individually using a single source of dLOS or OS for production of a single conjugate, and then different conjugates may be mixed subsequently to produce a vaccine containing more than one type of dLOS- or OS- carrier conjugate. In this way, a vaccine containing one or more of the known antigenic types of M. catarrhalis LOS may be produced.
  • dLOS Purified dLOS was characterized and compared to purified LOS using standard SDS-PAGE and silver staining techniques, substantially as described previously (Tsai, CM. & C.E. Frasch, 1982, Anal. Biochem. 119:115-119). Aliquots of M. catarrhalis LOS (25, 50, 100 and 200 ng) and dLOS (20 ⁇ g) were separated on the gel which also contained, as standards, Salmonella minnesota LPS Ra and Re. Each of the lanes containing M. catarrhalis LOS showed a single band of M r about 4,000 (Edebrink, P. et al., 1994, Carbohydr. Res.
  • LAL Limulus amebocyte lysate
  • a composition having a reduction of about 500-fold to about 1, 000- fold EU/ ⁇ g or more is used for a vaccine.
  • reductions of in vitro toxicity determined using, for example, the LAL assay correlate with reduced and acceptable levels of in vivo toxicity.
  • In vivo toxicity can be readily determined using standard in vivo pyrogen testing methods (e.g., in rabbits, using doses of 0.1 ⁇ g to 1 ⁇ g/kg of body weight).
  • the antigenicity of the dLOS, AH-dLOS and the dLOS-TT and dLOS-HMP conjugates was tested by double immunodiffusion using rabbit hyperimmune serum to M. catarrhalis whole cells (strain 25238).
  • the hyperimmune serum was prepared by standard methods. Briefly, two New Zealand white rabbits (female, 2 to 3 kg each) were injected subcutaneously and intramuscularly twice (both s.c. and i.m. for each injection) at four- week intervals with an emulsion of l ⁇ " M. catarrhalis whole cells (strain 25238) and incomplete Freund's adjuvant (at a ratio of 1:1, vol/vol). Blood samples were collected before and two weeks after each injection.
  • Double immunodiffusion was performed using standard methods in a 0.8 % agarose gel in phosphate-buffered saline (PBS, pH 7.4).
  • the central well contained the rabbit hyperimmune serum to M. catarrhalis whole cells and the surrounding wells individually contained LOS, dLOS-TT, dLOS-HMP, dLOS, and HMP.
  • the hyperimmune serum reacted with the LOS in the double-immunodiffusion assay, producing a sharp, readily detectable band of precipitation.
  • the hyperimmune serum reacted with the dLOS to produce a somewhat broader band of precipitation, showing that the isolated dLOS retained the antigenicity of the isolated LOS.
  • the hyperimmune serum also reacted with the dLOS-TT and dLOS-HMP conjugates, producing an identical band of precipitation when compared to LOS.
  • the hyperimmune serum did not react measurably with the isolated HMP.
  • Antigenicity was also measured using an enzyme linked immunosorbent assay (ELISA), using previously-described methods (Gu, X.X., et al., 1996, Infect. Immun. 64:4047-4053), with some modifications.
  • ELISA enzyme linked immunosorbent assay
  • the ELISA plates were coated with LOS and then blocked with 3 % BSA. Then, the ELISA wells were incubated with diluted rabbit serum, before alkaline phosphatase-conjugated goat anti-rabbit IgG and IgM (Sigma) was added. Between all of the steps, the wells were washed copiously with PBS containing a polymeric dispersing agent (0.01% Tween-20). The enzyme substrate was added for 30 min, and then the reactions were quantitated at A405.
  • the antigenicity of the dLOS-carrier conjugates was determined similarly, using the conjugates as coating antigens and a diluted rabbit immune serum as a binding antibody. Both dLOS-carrier conjugates showed comparable binding to rabbit hyperimmune serum, and the antigenicity of the dLOS-carrier conjugates was higher than that of LOS under the same conditions.
  • mice were injected parenterally into mice and rabbits and the levels of anti-LOS antibodies in the animals' sera was measured subsequently using ELISA.
  • a nonconjugated mixture of dLOS and TT or HMP did not elicit anti-LOS antibodies.
  • both dLOS-TT and dLOS-HMP conjugates elicited low levels of anti-LOS IgG after a second injection of the conjugate.
  • Both dLOS-TT and dLOS-HMP elicited similar levels of anti-LOS IgG after three injections.
  • LOS alone and the dLOS-carrier conjugates elicited similar levels of anti-LOS IgG.
  • Ribi-700 commercially available as Ribi-700, from Ribi Immunochemical Research, Hamilton, MT.
  • other well known standard adjuvants such as, for example, aluminum compounds (i.e. alum), chemically-modified lipopolysaccharide, suspensions of killed Bordetella pertussis, N-acetylmuramyl-L-alanyl- D-glutamine and other adjuvants known to one of ordinary skill in the art. Additional adjuvants are described by Warren et al. (Ann. Rev. Biochem. 4:369-388, 1986; New Generation Vaccines, 2nd Edition, Levine, M. M.
  • dLOS-TT elicited low level of IgG after the first injection, and that level rose significantly after the second and third injections.
  • Injection of the dLOS-TT conjugate with adjuvant enhanced the level of IgG produced in dLOS-TT injected group compared to the mice that received with the same conjugate without adjuvant.
  • the unconjugated mixture of TT and dLOS elicited higher levels of anti-TT IgG than that elicited by dLOS-TT. All immunogens elicited low levels of anti- TT IgM, which was increased by the inclusion of adjuvant in the injections.
  • Immunogenicity of the dLOS-protein conjugates was also determined for rabbits injected s.c. and i.m. at time 0 and one month later (injections were both s.c. and i.m. for each injections). Blood samples were collected at time 0 (i.e., at the first injection time), two weeks after the first injection, and two weeks after the second injection.
  • the levels of IgM and IgG were determined for rabbit sera obtained after injection with the following immunogens (all at 50 ⁇ g per immunogen per injection): LOS, dLOS-TT, dLOS-TT with adjuvant, dLOS-HMP, dLOS-HMP with adjuvant, an unconjugated admixture of dLOS, TT, and HMP, or whole M. catarrhalis cells.
  • immunogens all at 50 ⁇ g per immunogen per injection
  • LOS dLOS-TT
  • dLOS-TT with adjuvant dLOS-HMP
  • dLOS-HMP with adjuvant dLOS-HMP with adjuvant
  • an unconjugated admixture of dLOS, TT, and HMP or whole M. catarrhalis cells.
  • the mixture of dLOS, TT, and HMP or LOS alone elicited low levels of anti-LOS IgG or IgM antibodies after two injections.
  • the dLOS-TT conjugate elicited a significant rise of anti-LOS IgG after the first and second injections compared to pre-injection serum levels. Injection of the dLOS-HMP conjugate elicited lower levels of IgG than did the dLOS-TT conjugate. Inclusion of an adjuvant enhanced the levels of anti-LOS IgG for both conjugates after each injection, and there was no significant difference between the two conjugates after two injections with adjuvant.
  • both conjugates elicited low to medium levels of anti-LOS antibodies, and inclusion of an adjuvant elicited generally increased levels of anti-LOS IgM antibodies detected after each injection, compared to the same conjugate injected without adjuvant.
  • mice and rabbits The antisera produced in mice and rabbits was assayed for bactericidal activity in vitro against homologous and heterologous strains of M. catarrhalis, using standard methods (Gu X-X., et al., 1996, Infect. Immun. 64:4047-4053).
  • rabbit model sera produced after immunization with LOS or unconjugated dLOS showed no bactericidal activity against the homologous M. catarrhalis strain.
  • the anti-LOS IgG levels as determined by ELISA, conelated with the detected bactericidal titers.
  • the bactericidal activities of the antisera against the homologous M. catarrhalis strain and heterologous strains from different geographic areas (e.g., Japan) showed that rabbit sera produced in response to the dLOS-protein conjugates had more bactericidal activity than did similarly produced mouse sera. All of the conjugate-induced rabbit sera showed bactericidal activity against the homologous M. catarrhalis strain and representative sera showed bactericidal activity against most nonhomologous strains (9 of 10 ATCC strains and clinical isolates). In contrast, less than half of the dLOS-carrier conjugate-induced mouse antisera showed bactericidal activities against the homologous strain. Generally, the bactericidal titers and the levels of anti-LOS IgG antibody correlated.
  • the bactericidal activities of the rabbit antisera elicited by dLOS-TT formulated with an adjuvant were analyzed using twenty additional M. catarrhalis strains (ten wild type ATCC strains and ten clinical isolates). Ten of twenty strains were either complement sensitive or serum sensitive. Using the remaining ten strains, the rabbit antisera demonstrated bactericidal activities to four ATCC and five clinical isolates at the mean titer of 1:15 (range 1:2 to 1:32). One strain was negative in the bactericidal assay.
  • mice In the mouse model, 20% (4 of 20 mice) of sera from mice immunized with the dLOS-protein conjugates, and 45% (9 of 20 mice) of sera produced after immunization with conjugates and adjuvant, showed low titers of bactericidal activity against the homologous M. catarrhalis strain (ATCC 25238) after three injections of dLOS-carrier conjugate.
  • the dLOS component become immunogenic. That is, the M. catarrhalis dLOS-carrier conjugates induced significant IgG antibody responses to LOS in mammals. In mammals, the dLOS-carrier conjugates elicited at least similar levels of anti-LOS IgG antibodies as did the LOS.
  • the immunogenicity of the dLOS-protein conjugates was better in rabbits than in mice (i.e., after two injections of the conjugates into rabbits, the fold increase of anti-LOS antibodies was generally higher than the fold increase of anti-LOS antibodies seen in mice after two or three injections of the same conjugates).
  • the levels of anti-LOS antibodies were enhanced when the conjugate was injected with adjuvant compared to injection of the same conjugate without adjuvant.
  • Both the dLOS-TT and the dLOS-HMP conjugates elicited similar levels of anti-LOS IgG antibodies, which were increased when the conjugates were formulated with an adjuvant.
  • the M. catarrhalis dLOS-carrier conjugates of the present invention are useful as a vaccine to induce immunity against M. catarrhalis infections in mammals, particularly for preventing otitis media and respiratory diseases in humans.
  • the methods of producing such dLOS-carrier conjugates as disclosed herein are useful for the manufacturing of such vaccines.
  • the methods disclosed herein are also useful for identifying other dLOS-carrier conjugates (i.e., conjugates of dLOS with other carrier moieties) that are useful for inducing protective immune responses to M. catarrhalis in mammals, particularly in humans, including children. It will be understood that a vaccine against M.
  • catarrhalis may include dLOS-carrier conjugate, along with other components, such as immunogenically inert pharmaceutically acceptable agents or clinically acceptable adjuvant.
  • M. catarrhalis dLOS- carrier conjugate may also be combined with other immunogenically active components directed against other infectious agents (e.g., to produce a combination vaccine against M. catarrhalis and one or more other bacteria or virus that causes childhood disease); for example, a trivalent vaccine against M. catarrhalis, nontypeable Haemphilus influenzae and Streptococcus pneumoniae to prevent bacterial otitis media.
  • the dLOS-carrier conjugates are parenterally administered.
  • various routes of vaccine administration including, for example, intramuscular (i.m.), subcutaneous (s.c), intraperitoneal (i.p.), transmucosal (e.g., intranasally) and intraarterial are contemplated, transmucosal, s.c. and i.m. administration are preferred.
  • the dLOS-carrier conjugates may be in the form of a sterile preparation, such as, for example, a sterile injectable aqueous or oleaginous suspension, with or without an adjuvant.
  • the sterile preparation may also be a sterile injectable solution or suspension in a parenterally acceptable diluent or solvent, such as, for example, a solution in 1,3-butanediol.
  • a parenterally acceptable diluent or solvent such as, for example, a solution in 1,3-butanediol.
  • suitable diluents include, for example, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils may be employed conventionally as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may likewise be used in the preparation of injectable preparations.
  • the formulation may be aerosolized using an inert carrier (e.g., air or hydrocarbon) using any of a variety of conventional methods.
  • the dLOS-carrier conjugates in a vaccine of the present invention may be in soluble or microparticular form, or may be incorporated into microspheres or microvesicles, including liposomes.
  • the dosage of the conjugate administered will range from about 10 ⁇ g to about 100 ⁇ g, preferably, between about 20 ⁇ g and about 50 ⁇ g. In another preferred embodiment, the amount administered is about 25 ⁇ g to about 40 ⁇ g.
  • the exact dosage can be determined by routine dose/response protocols known to one of ordinary skill in the art, generally with doses administered on the basis of body weight, particularly for children.
  • the vaccine of the invention may be administered to mammals of any age and are adapted to induce active immunization in young mammals, particularly humans, against otitis media and respiratory infections caused by M. catarrhalis.
  • the dLOS-carrier conjugate is administered at about two to twelve months of age, preferably between about two to six months of age.
  • Booster injections will likely be given.
  • two booster injections of between about 10 ⁇ g and about 25 ⁇ g are administered, for example, at about two months and about thirteen months after the initial injection.
  • booster injections are given at two, four and sixteen months after the initial injection.
  • Other booster injection protocols are also contemplated.
  • Vaccine compositions may comprise a cocktail of conjugates from different M. catarrhalis strains that protects against all or most medically relevant strains.
  • M. catarrhalis strains There are three known types of M. catarrhalis based on dLOS: Types A, B and C which represent 61%, 29% and 5% of clinical isolates, respectively. As shown in Example 6, antisera raised against one strain cross-reacts with some, but not all, other strains. Thus, a cocktail of different conjugates will likely be used. Mixtures of conjugates containing dLOS or OS from Types A and B will cover 90% of all medically relevant strains, while mixtures of conjugates containing dLOS or OS from Types A, B and C will cover 95% of all medically relevant strains.
  • This mouse pulmonary clearance model mimics the natural transmission of the bacteria in humans. The advantages of this model are that it is simple, repeatable and well controlled by the aerosol machine, large numbers of mice can be studied under the same challenge conditions and there is no surgically invasive procedure required for the inoculation of bacteria.
  • bactericidal antibodies elicited in response to the dLOS-carrier conjugates, particularly
  • IgG may transude to mucosal surfaces of nasopharynges. There, the antibodies can inactivate a M. catarrhalis innoculum on the mucosal surface, thus preventing or relieving symptoms of M. catarrhalis-caxxsc ⁇ otitis media and respiratory diseases.
  • Secretory IgA may also play a role in respiratory mucosal immunity, particularly if the conjugate vaccine is administered to the nasal mucosa.
  • Example 1 Purification and Detoxification of LOS from M. catarrhalis M. catarrhalis (type A) ATCC strain 25238 was used as an exemplary source for purification of LOS (Edebrink, P., et al., 1994, Carbohydr. Res. 257:269-284; Masoud, H., et al., 1994, Can. J. Chem. 72:1466-1477). The strain was grown on chocolate agar at 37°C, 5% CO 2 , for 8 hr, and transferred to 250 mL of 3% tryptic soy broth (TSB) (Difco Laboratories, Detroit, Mich.) in a 500-mL bottle.
  • TTB tryptic soy broth
  • the bottle was incubated at 110 rpm in an incubator shaker (Model G-25, New Brunswick Scientific, Co., Edison, NJ) at 37°C overnight.
  • the culture was transferred to six 2.8-liter baffled Fernbach flasks, each of which contained 1.4 liters of TSB.
  • the flasks were shaken at 110 rpm and maintained at 37°C for 24 hr.
  • the culture was centrifuged at 15,000 x g, at 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 using conventional methods (substantially as described in Masoud, H., et al., 1994, Can. J. Chem. 72:1466-1477), and dried to a powder.
  • the conventional methods substantially as described in Masoud, H., et al., 1994, Can. J. Chem. 72:1466-1477, and dried to a powder.
  • LOS was extracted from cells by a standard hot phenol-water method (Westphal, O. et al., 1965, Methods Carbohydr. Chem. 5:83-91) with modifications (Gu X-X., 1995, Infect. Immun. 63:4115-4120), which yields LOS with a protein and nucleic acid content of less than 1% (Smith, P.K., et al., 1985, Anal. Biochem. 150:76-85; Warburg, O. & W. Christian, 1942, Biochem. Z. 310:384-421).
  • Anhydrous hydrazine treatment of LOS using mild alkali conditions were used to remove 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 hr with mixing. This suspension was cooled on ice and cold acetone is added dropwise until a precipitate formed. The mixture was centrifuged at 5,000 x g, at 5°C for 30 min.
  • the pellet was washed twice with cold acetone, dissolved in pyrogen-free water at a final concentration of 10-20 mg/mL, and then ultracentrifuged at 150,000 x g, at 5°C for 3 hr.
  • 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 a micro phenol-sulfuric acid method (Dubois, M., et al., 1956, Anal. Biochem. 28:250-256).
  • the carbohydrate-containing fractions were pooled, freeze-dried, and designated as dLOS which was about 38 % of LOS by weight.
  • This method for detoxification of M catarrhalis LOS resulted in a better yield of dLOS after conjugating to protein carriers, compared to mild-acid treatment of LOS to cleave the lipid A portion from the LOS molecule at the Kdo-glucosamine linkage (i.e., the method of Gu, X.X., & CM. Tsai, 1993, Infect. Immun. 61:1873-1880).
  • Adipic hydrazide (AH) derivatives of dLOS prepared according to the methods of Example 1, were made and purified as follows.
  • Adipic acid dihydrazide (ADH) Aldrich Chemical Co., Milwaukee, Wis.
  • ADH Adipic acid dihydrazide
  • EDC l-ethyl-3-(3- dimethylaminopropyl) carbodiimide HC1
  • Sulfo-NHS N-hydroxysulfo-succinimide
  • dLOS 70 mg was dissolved in 7 mL of 345 mM ADH (molar ratio of ADH to LOS is aboutl00:l, based on an estimated 3,000 M r for dLOS) (Edebrink, P., et al., 1994, Carbohydr. Res. 257:269-284).
  • Sulfo-NHS was added to a concentration of 8 mM, the pH adjusted to 4.8, and EDC added to a concentration of 0.1 M.
  • the reaction mixture was stined and maintained at pH 4.8 for 3 hr.
  • the reaction mixture was adjusted to pH 7.0 and passed through the G-50 column as described in Example 1.
  • AH-dLOS was measured for its composition using dLOS and ADH as standards.
  • the AH-dLOS was conjugated to proteins (TT and HMP) as follows. TT (Connaught Labs. Inc., Swiftwater, Pa.) and HMP was purified from nontypeable Haemophilus influenzae strain 12 (Barenkamp, S.J., 1996, Infect. Immun.
  • AH-dLOS was coupled to TT or HMP to form conjugates (Gu, X.X., & CM. Tsai, 1993, Infect Immun. 61:1873-1880). Briefly, AH-dLOS (30 mg) was dissolved with 3 mL 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 of 150K) and HMP (M r of 120K) was aboutl00:l. The pH was adjusted to 5.4 and EDC added to a concentration of about 0.05 to 0.1 M.
  • the reaction mixture was stirred and the pH was maintained at 5.6 for 1 hr to 3 hr.
  • 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 as dLOS-TT or dLOS-HMP. Both conjugates were analyzed for their composition of carbohydrate and protein using dLOS and BSA as standards (Dubois, M., et al., 1956, Anal. Biochem. 28:250-256; Smith, P.K., et al., 1985, Anal. Biochem. 150:76-85).
  • the derivatized AH-dLOS and dLOS-protein conjugates were physically characterized based on the measured amounts ( ⁇ g/ml) of AH and dLOS in the derivatized product, or dLOS and protein in the conjugates.
  • dLOS-TT 103 ⁇ g/ml of dLOS and 266 ⁇ g/ml of protein were measured; and for dLOS-HMP, 220 ⁇ g/ml of dLOS and 280 ⁇ g/ml of protein were measured.
  • the molar ratios for the conjugates were calculated as moles of dLOS per mole of protein, using molecular weights of 3,000 for dLOS, 150,000 for TT, and 12,000 for HMP.
  • the molar ratios of dLOS to TT and to HMP in two conjugate preparations were 19:1 and 31:1, respectively.
  • the yields for the conjugates were calculated based on the starting amount of dLOS and the dLOS contained in the conjugates as measured by the phenol-sulfuric acid method. The yields were 8% for dLOS-TT and 19% for dLOS-HMP.
  • Example 3 Antigenicity of dLOS. Derivatized dLOS and dLOS-Protein
  • Double immunodiffusion was performed using standard methods in a 0.8 % agarose gel in phosphate-buffered saline (PBS, pH 7.4).
  • the central well contained the rabbit hyperimmune serum to M. catarrhalis whole cells and the surrounding wells individually contained the following: 1 mg/ml of LOS, 103 ⁇ g/ml of dLOS-TT, 220 ⁇ g/ml of dLOS-HMP (based on the amount of dLOS), 1 mg/ml of dLOS, 200 ⁇ g/ml of dLOS, and 500 ⁇ g/ml of HMP.
  • the two conjugates and the LOS formed substantially identical precipitation lines by double- immunodiffusion.
  • the hyperimmune serum did not react measurably with the isolated HMP.
  • ELISA was performed substantially as described previously (Gu, X.X., et al., 1996, Infect. Immun. 64:4047-4053), with the following modifications. After LOS (at 10 ⁇ g/ml) coating of the wells of a standard ELISA plate (Dynatech Laboratories, Inc.,
  • Example 4 Immunogenicity of dLOS-Protein Conjugates in Mice
  • the adjuvant used contained 50 ⁇ g of monophosphoryl lipid A (MPL) and 50 ⁇ g of synthetic trehalose dicorynomycolate (STD) per injection, in an inert carrier (commercially available as Ribi-700, from Ribi ImmunoChem research, Inc., Hamilton, Mt).
  • MPL monophosphoryl lipid A
  • STD synthetic trehalose dicorynomycolate
  • Serum anti-LOS levels were expressed as ELISA units (EU), using LOS isolated from the 25238 strain as a coating antigen.
  • EU ELISA units
  • hyperimmune serum to whole cells of the 25238 strain was used and assigned values of 65,000 EU/mL for IgG and 800 EU/mL for IgM.
  • Serum antibodies against TT or HMP were measured by
  • ELISA in which TT or HMP (5 ⁇ g/mL) was used as a coating antigen and expressed as ELISA units on the basis of a reference mouse serum (produced by three injections of TT or HMP), which was assigned values of 2,000 EU/mL for IgG and 10 EU/mL for IgM.
  • antibody levels are expressed as the geometric mean ELISA units or titers (reciprocal) of n independent observations ⁇ standard deviation or range (n ⁇ 4). Significance was tested with the two-sided t test and P values smaller than 0.05 were considered significant.
  • mice for each group were given a total of three subcutaneous injections at three-week intervals with 5 ⁇ g of LOS, 5 ⁇ g of conjugates, 5 ⁇ g of conjugates with
  • the ELISA units were based on a reference serum against strain 25238, and the LOS from strain 25238 was used as a coating antigen. For data marked with * and **for a single immunogen, the measurements are significantly different (p ⁇ 0.01). Anti-protein antibodies in mice. As shown by the data presented in Table 2, dLOS-TT elicited low levels of anti-TT IgG antibodies after the first injection, and the levels rose significantly after the second and third injections (P ⁇ 0.01). Injection with the Ribi adjuvant enhanced the level of IgG in dLOS-TT group. The mixture of TT and dLOS elicited higher level of IgG than that elicited by dLOS-TT. All immunogens elicited low levels of anti-TT IgM.
  • Table 2 Also shown in Table 2 are the results obtained for anti-HMP antibodies.
  • the dLOS-HMP conjugate elicited low level of IgG after the first injection, that level rose significantly after the second and third injections (P ⁇ 0.01).
  • the Ribi adjuvant enhanced the levels of IgG in dLOS-HMP group.
  • the mixture of HMP and dLOS elicited higher level of IgG than that of dLOS-HMP. All immunogens elicited low levels of anti-HMP IgM.
  • mice for each group were given a total of three subcutaneous injections at three-week intervals with 5 ⁇ g of LOS, 5 ⁇ g of conjugates, 5 ⁇ g of conjugates with Ribi adjuvant, or a mixture of dLOS and TT or HMP (5 ⁇ g each).
  • Rabbits (two or three per group) were injected individually two times (s.c. and i.m.) at four-week intervals with 50 ⁇ g/injection of: LOS, the conjugate dLOS-TT with or without adjuvant, the conjugate dLOS-HMP with or without adjuvant, or an admixture of dLOS plus TT or HMP (50 ⁇ g of each component).
  • the immunogen was in 1 mL of 0.9 % NaCl.
  • the immunogen was in 1 mL of 0.9 % NaCl containing 250 ⁇ g of monophosphoryl lipid A and 250 ⁇ g of trehalose dimycolate (Ribi-700 adjuvant, Ribi Immunochemical Research, Hamilton, MT). At two weeks after each injection, 10-20 ml blood samples were collected from an ear vein using standard procedures.
  • the admixture of dLOS, TT, and HMP, or LOS elicited low levels of anti-LOS IgG or IgM antibodies after two injections.
  • the dLOS-TT conjugate elicited a significant rise of anti-LOS IgG after the first and second injections (37- and 700-fold above the pre-immunization sera).
  • the dLOS-HMP showed lower levels of IgG than dLOS-TT (6- and 347-fold, respectively, above the pre-immunization sera levels).
  • the Ribi adjuvant enhanced the levels of anti- LOS IgG in both conjugate groups after each injection (40- to 2,000-fold above the pre- immunization sera levels).
  • D Blood samples were collected at: 0, before injection of immunogen; 1, at 14 days after the first injection; and 2, at 14 days after the second injections.
  • Anti-protein antibodies in rabbits As shown by the data for anti-TT antibodies presented in Table 4, dLOS-TT elicited significant level of IgG after two injections (389-fold above the pre-immunization sera level).
  • the Ribi adjuvant enhanced the levels of IgG elicited by dLOS-TT by 4-fold after two injections.
  • the mixture of TT and dLOS elicited a higher level of anti-TT IgG than did the dLOS-TT conjugate, especially after one injection. All immunogens elicited low levels of anti-TT IgM.
  • dLOS-HMP elicited significant level of IgG after two injections (81 -fold above pre-immunization sera).
  • Inclusion of the Ribi adjuvant enhanced the levels of IgG elicited by dLOS-HMP by 4-fold after two injections.
  • the mixture of HMP and dLOS elicited a higher level of IgG than that of dLOS-HMP, especially after one injection. All immunogens elicited low levels of anti- HMP IgM.
  • Example 6 Bactericidal Activity of Animal Sera against M. catarrhalis Strains
  • bactericidal activity of the animal sera prepared according to Examples 4 and 5 was tested against the same M. catarrhalis strain from which the LOS had been isolated (ATCC 25238; "homologous strain") and against other wild-type strains of M. catarrhalis ("heterologous strains). Eleven wild type strains of M. catarrhalis ATCC Nos.
  • bactericidal assay For the bactericidal assay, rabbit pre- and post-immune sera (after two injections) were inactivated for complement components by incubating at 56°C for 30 min. The inactivated sera were then tested for bactericidal activity against M. catarrhalis strains using a complement-mediated bactericidal assay substantially as described previously (Gu, X.X., et al., 1996, Infect. Immun. 64:4047-4053), except that a guinea pig serum (1:1 dilution, 20 ⁇ l per well) was used as a source of complement (Sigma, St. Louis, MO) and the reaction plate was incubated at 37°C for 30 min before plating into agar plates. The highest serum dilution that caused more than 50% killing was expressed as the reciprocal bactericidal titer.
  • the bactericidal activities of the rabbit antisera elicited by dLOS-TT formulated with Ribi adjuvant were further analyzed using ten additional wild type strains (ATCC strains) and ten of the Japanese clinical isolates. Ten of twenty strains were either complement sensitive (strains 23246, 43617, M9) or serum sensitive (strains 43627, 43628, 49143, M4, M7, M8, M10). Using the remaining ten strains, the rabbit antisera demonstrated bactericidal activities to four ATCC and five Japanese strains at the mean titer of 1:15 (1:2 to 1:32). One strain (ATCC 25240) was negative in the bactericidal assay.
  • Example 7 Passive protection study in mouse pulmonary clearance model Forty mice were immunized with either rabbit antisera against dLOS-TT, or pre- immune sera, and then challenged with 10 cfu of M. catarrhalis strain 25238 by aerosol chamber 18 hours after the immunization. The mice were sacrificed at 3 and 6 hours after the challenge (Fig. 2). The lungs and blood samples were collected for analysis. The results are shown in Fig. 3. At three hours post-challenge, the amount of bacteria in the vaccine group was reduced by 50% compared to the control. There was a
  • dLOS-carrier conjugates prepared as described in Examples 1 and 2, or OS-carrier conjugates, prepared as described in Examples 9-11.
  • Individuals are screened for relatively low levels of endogenous antibody (e.g., resulting from childhood infections with M. catarrhalis) and adults with relatively low levels compared to the general population are chosen for the study.
  • These individuals are intramuscularly injected with either the dLOS-TT conjugate, the dLOS-HMP conjugate, the OS-TT conjugate or the OS-HMP conjugate (25 ⁇ g to 50 ⁇ g, depending on body weight) in a pharmaceutically acceptable carrier.
  • one injection is generally sufficient to elicit an antibody response within three days to two weeks.
  • Immunogenicity and bactericidal activity of the resulting antisera are determined using methods substantially as described in Examples 4-6.
  • a second injection is administered about one to six months after the first injection and the level of serum anti- M. catarrhalis antibodies is measured about one week later.
  • Control individuals are injected with a control vaccine of the same amount of the corresponding protein component of the conjugate (alone) in the same pharmaceutically acceptable carrier, and on the same injection schedule as for immunized adults.
  • serum antibody levels after immunization show that the conjugates are immunogenic in vivo without producing unacceptable side effects.
  • Bactericidal activity is associated with the serum antibodies from these individuals.
  • multiple immunizations are preferred to achieve optimal antibody response.
  • serum obtained from control individuals who receive control injections exhibit no measurable immunogenicity or anti- M. catarrhalis bactericidal activity above that found in their pre-injection sera. That is, antisera from adults who receive the dLOS-TT conjugate, dLOS-HMP conjugate, OS-TT conjugate or OS-HMP conjugate exhibit significantly more immunogenicity or anti-M catarrhalis bactericidal activity compared to the control group.
  • the frequency of occunence of middle ear infections in the individuals is monitored over several years and none of the adults immunized with the dLOS-TT, dLOS-HMP, OS-TT or OS-HMP conjugate experiences a middle ear infection during that time.
  • testing of the vaccine is extended to children who receive an initial dose of vaccine i.m. (amounts based on body weight, generally 10-40 ⁇ g) at between two and four months of age, and two booster vaccinations at two and four months after the initial vaccination.
  • One group of children receives three booster vaccinations administered at two, four months and sixteen months after the initial vaccination.
  • Age-matched children who did not receive the vaccine are used as control subjects.
  • Serum antibodies are monitored in the immunized children and detected as described above, indicating both immunogenicity and bactericidal activity. The children are monitored for otitis media and sinusitis from after the first vaccination until about four years of age. Children that receive the vaccinations have significantly fewer episodes of otitis media and sinusitis, and diminished symptoms when otitis media and/or sinusitis is detected, during the monitoring period compared to control subjects.
  • Example 9 Acid hydrolysis of M. catarrhalis strain 25238 LOS to produce oligosaccharide (OS ⁇ M. catarrhalis LOS was detoxified by mild-acid treatment of LOS to cleave the lipid A portion from the LOS molecule at the Kdo-glucosamine linkage (i.e., the method of Gu, X.X., & CM. Tsai, 1993, Infect. Immun. 61:1873-1880) to produce oligosaccharide (OS). Briefly, 421 mg of LOS was dissolved in distilled water to 10 mg/ml, then hydrolyzed in 1% acetic acid at 100°C for 2 to 3 hours.
  • ADH was coupled to OS by carbodiimide-mediated condensation with EDC and sulfo-NHS as described previously (Gu et al., 1993, supra.).
  • the reaction mixture was purified with a Bio-Gel P-4 column (1.6 x 90 cm; Bio-Rad, Hercules, CA).
  • the eluate was assayed for sugar and AH content. Peaks containing both sugar and AH were pooled and lyophilized three times to remove the salt.
  • the ratio of OS to ADH was around 1.
  • the yield of sugar in AH-OS derivative was about 74%.
  • Example 11 Conjugation of AH-OS to proteins The coupling reaction was performed at pH 5.2 ⁇ 0.2 with 0.05 - 0.1 M EDC.
  • OS-TT reaction 30 mg of AH-OS was dissolved in 3 ml distilled water and mixed with 15 mg of TT (5.90 mg/ml).
  • OS-HMP reaction 20 mg of AH-OS was dissolved in 2 ml water and mixed with 8.4 mg of HMP (4.2 mg/ml).
  • the molar ratio of AH-OS to TT or HMP was 150 or 141 to 1 (based on molecular weights: OS as 2,000, TT as 150,000, and HMP as 120,000).
  • the purification steps were the same as for the dLOS-protein conjugates described in Example 2.
  • OS-protein conjugates have the same utility as the dLOS-protein conjugates discusses above, namely as a vaccine for Moraxella (Branhamella) catarrhalis infections in humans.
  • the composition, yield and antigenicity of the OS-TT and OS-HMP conjugates are shown in Table 5.
  • the ratio is expressed as moles of OS per mole of protein with molecular weights of
  • the OS-protein conjugates elicited antibody responses in both mice and rabbits against LOS.
  • the protein conjugates also elicit antibodies against TT and HMP in both mice and rabbits.
  • the murine antibody response to M. catarrhalis strain 25238 LOS elicited by conjugates is shown in Table 6.
  • the adjuvant used was the same as for the dLOS antibody response.
  • the injections were given three times at three-week intervals and the mice were bled fourteen days after the first injection and seven days after the second and the third injections.
  • Serum anti-LOS levels were expressed as ELISA units (EU), using LOS isolated from the 25238 strain as a coating antigen.
  • EU ELISA units
  • hyperimmune serum to whole cells of the 25238 strain was used and assigned values of 65,000 EU/mL for IgG and 800 EU/mL for IgM.
  • Serum antibodies against TT or HMP were measured by ELISA as described for the dLOS conjugates. For statistical analysis of these results, antibody levels are expressed as the geometric mean ELISA units or titers (reciprocal) of n independent observations ⁇ standard deviation or range (n ⁇ 4). Significance was tested with the two-sided t test and P values smaller than 0.05 were considered significant.
  • OS-TT1 1 2 (1-3) 3 (1-9) + adjuvant 2 4 (1-22) 3 (1-9) 3 7 (1-48) 50 (12-209)
  • mice for each group were given a total of three subcutaneous injections at 3 -week intervals with 5 ug of conjugates, conjugates with Ribi adjuvant, LOS, or the mixture of OS, TT, and HMP (5 ug each). Blood samples were collected 2 weeks after the 1st injection, 1 week after the 2nd and 3rd injection.
  • the ELISA units were based on a reference serum against strain 25238, and the LOS from strain 25238 was used as a coating antigen.
  • Pre-immune sera contained 1 (1-2) U of IgG and 1 U of IgM.
  • the murine antibody response to TT elicited by OS-TT conjugates is shown in Table 7.
  • the IgG antibody response was much stronger than the IgM antibody response.
  • the adjuvant significantly enhance the immune response for OS-TT2, but did not have as dramatic an effect for OS-TTl .
  • OS-TT2 1 113 (66-191) 3 (1-5) + adjuvant 2 271 (81-908) 19 (9-41) 3 1,756 (454-6,769) 90 (48-169)
  • OS-HMPl 1 9 (2-34) 2 (1-3) + adjuvant 2 585 (330-1,442) 5 (3-8) 3 1,506 (507-4,464) 6 (3-12)
  • OS-HMP2 1 81 (45-146) 6 (3-14) + adjuvant 2 1,131 (642-1,997) 17 (8-38) 3 4,228 (2,600-7,454) 38 (12-120)
  • the ELISA units were based on a reference serum against HMP, and HMP was used as a coating antigen. Pre-immune sera showed 1 to 3 unit of IgG or IgM.
  • the rabbit antibody responses to M. catarrhalis LOS elicited by conjugates is shown in Table 9, and the rabbit antibody response to TT or HMP elicited by conjugates is shown in Table 10. TABLE 9. Rabbit antibody response to M. catarrhalis LOS elicited by conjugate
  • OS-TT2+Ribi 0 4 (3-10) 10 (3-30) 1 729 (270-2,430) 156 (90-270) 2 12,627 (7,290-21,870) 90
  • OS-HMP2 0 17 (10-30) 7 (3-30) 1 467 (90-2,430) 10 (3-30)
  • OS-TT2+Ribi 0 17 (10-30) 10 1 2,430 90 2 21,870 355 (90-810)
  • OS-HMP2+Ribi 0 10-30) 1 468 (270-810) 30

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Abstract

L'invention concerne un vaccin conjugué contre Moraxella (Branhamella) catarrhalis comprenant un lipooligosaccharide isolé dont on retiré des acides gras estérifiés pour produire un lipooligosaccharide détoxiqué (dLOS), ou dont on a retiré un lipide A pour produire un oligosaccharide détoxiqué (OS), qui est lié à un excipient immunogène. Le vaccin s'utilise pour la prévention de l'otite moyenne et des infections respiratoires provoquées par M. catarrhalis chez des mammaliens, y compris des humains.
PCT/US1999/000590 1996-04-23 1999-01-12 Vaccin a base de lipooligosaccharide destine a la prevention d'infections dues a moraxella (branhamella) catarrhalis chez les mammaliens WO1999036086A1 (fr)

Priority Applications (12)

Application Number Priority Date Filing Date Title
CA002315746A CA2315746A1 (fr) 1998-01-13 1999-01-12 Vaccin a base de lipooligosaccharide destine a la prevention d'infections dues a moraxella (branhamella) catarrhalis chez les mammaliens
BR9906902-4A BR9906902A (pt) 1998-01-13 1999-01-12 "vacina de conjugado para moraxella catarrhalis, composição farmacêutica, lipooligossacarìdeo, e, processos de prevenção de otite média causada pela infecção com moraxella catarrhalis em um mamìfero, de desintoxicação de lipooligossacarìdeo (los) isolado de moraxella catarrhalis, e de preparação de uma vacina de conjugado contra moraxella catarrhalis"
IL13690399A IL136903A0 (en) 1998-01-13 1999-01-12 Lipooligosaccharide-based vaccine for prevention of moraxella (branhamella) catarrhalis infections in mammals
JP2000539859A JP2002509115A (ja) 1998-01-13 1999-01-12 哺乳動物におけるモラクセラ（ブランハメラ）カタラリス感染の予防のためのリポオリゴ糖ベースのワクチン
KR1020007007737A KR20010034124A (ko) 1998-01-13 1999-01-12 포유동물에서 모락셀라(브란하멜라) 카타르할리스 감염의예방을 위한 리포올리고당에 기초한 백신
AU22212/99A AU2221299A (en) 1998-01-13 1999-01-12 Lipooligosaccharide-based vaccine for prevention of (moraxella) ((branhamella)) (catarrhalis) infections in mammals
EP99902170A EP1047447A1 (fr) 1998-01-13 1999-01-12 VACCIN A BASE DE LIPOOLIGOSACCHARIDE DESTINE A LA PREVENTION D'INFECTIONS DUES A $i(MORAXELLA) $i((BRANHAMELLA)) $i(CATARRHALIS) CHEZ LES MAMMALIENS
US09/610,034 US6685949B1 (en) 1998-01-13 2000-07-05 Lipooligosaccharide based vaccine for prevention of moraxella (branhamella)catarrhalis infections in humans
AU2003200964A AU2003200964B2 (en) 1998-01-13 2003-02-21 Lipooligosaccharide-Based Vaccine for Prevention of Moraxella (Branhamella) Catarrhalis Infections in Mammals
US10/688,115 US20040126381A1 (en) 1996-04-23 2003-10-17 Intranasal immunization with detoxified lipooligosaccharide from nontypeable haemophilus influenzae or moraxella
US10/729,027 US20040115214A1 (en) 1998-01-13 2003-12-05 Lipooligosaccharide-based vaccine for prevention of Moraxella (Branhamella) catarrhalis infections in humans
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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US60/071,483 1998-01-13

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US09/789,017 Continuation US6607725B2 (en) 1996-04-23 2001-02-20 Conjugate vaccine for nontypeable Haemophilus influenzae

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002089839A1 (fr) * 2001-05-03 2002-11-14 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Immunisation intranasale par un lipooligosaccharide detoxifie provenant d'un haemophilus influenzae ou d'un moraxella catarrhalis non typable
WO2006085910A2 (fr) * 2004-06-04 2006-08-17 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Moraxella catarrhalis exempte d'endotoxine
EP1880734A3 (fr) * 1999-07-15 2008-06-25 Sanofi Pasteur Limited Composition immunogène multivalente servant a conférer une protection chez un hôte contre une maladie provoquée a Ia fois par Haemophilus influenzae et par Moraxella catarrhalis
US7641906B2 (en) 1996-04-23 2010-01-05 The United States Of America As Represented By The Department Of Health And Human Services Intranasal immunization with detoxified lipooligosaccharide from nontypeable Haemophilus influenzae or Moraxella catarrhalis

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104592365B (zh) * 2013-11-01 2019-06-14 中国人民解放军军事医学科学院毒物药物研究所 梭曼抗原、梭曼疫苗及其用途
EP3706787A4 (fr) * 2017-11-06 2021-10-20 OZQ Pty Ltd Vaccin

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998053851A1 (fr) * 1997-05-28 1998-12-03 University Of Iowa Research Foundation Mutants laft de bacteries pathogenes gram negatif

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998053851A1 (fr) * 1997-05-28 1998-12-03 University Of Iowa Research Foundation Mutants laft de bacteries pathogenes gram negatif

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
EDEBRINK, PER ET AL: "Structural studies of the O-antigen oligosaccharides from two strains of Moraxella catarrhalis serotype C", CARBOHYDR. RES. (1995), 266(2), 237-61 CODEN: CRBRAT;ISSN: 0008-6215, XP004022196 *
GU X X ET AL: "Preparation, characterization, and immunogenicity of meningococcal lipooligosaccharide-derived oligosaccharide-protein conjugates.", INFECTION AND IMMUNITY, (1993 MAY) 61 (5) 1873-80. JOURNAL CODE: GO7. ISSN: 0019-9567., United States, XP002102900 *
GU X X ET AL: "Synthesis and characterization of lipooligosaccharide-based conjugates as vaccine candidates for Moraxella (Branhamella) catarrhalis.", INFECTION AND IMMUNITY, (1998 MAY) 66 (5) 1891-7. JOURNAL CODE: GO7. ISSN: 0019-9567., United States, XP002102901 *
GU X X ET AL: "Synthesis, characterization, and immunologic properties of detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins.", INFECTION AND IMMUNITY, (1996 OCT) 64 (10) 4047-53. JOURNAL CODE: GO7. ISSN: 0019-9567., United States, XP002102897 *
KELLY J ET AL: "Separation and characterization of O-deacylated lipooligosaccharides and glycans derived from Moraxella catarrhalis using capillary electrophoresis-electrospray mass spectrometry and tandem mass spectrometry.", ANALYTICAL BIOCHEMISTRY, (1996 JAN 1) 233 (1) 15-30. JOURNAL CODE: 4NK. ISSN: 0003-2697., United States, XP002102899 *
MURPHY T F: "Branhamella catarrhalis: epidemiology, surface antigenic structure, and immune response.", MICROBIOLOGICAL REVIEWS, (1996 JUN) 60 (2) 267-79. REF: 186 JOURNAL CODE: M9M. ISSN: 0146-0749., United States, XP002102898 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7641906B2 (en) 1996-04-23 2010-01-05 The United States Of America As Represented By The Department Of Health And Human Services Intranasal immunization with detoxified lipooligosaccharide from nontypeable Haemophilus influenzae or Moraxella catarrhalis
EP1880734A3 (fr) * 1999-07-15 2008-06-25 Sanofi Pasteur Limited Composition immunogène multivalente servant a conférer une protection chez un hôte contre une maladie provoquée a Ia fois par Haemophilus influenzae et par Moraxella catarrhalis
WO2002089839A1 (fr) * 2001-05-03 2002-11-14 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Immunisation intranasale par un lipooligosaccharide detoxifie provenant d'un haemophilus influenzae ou d'un moraxella catarrhalis non typable
WO2006085910A2 (fr) * 2004-06-04 2006-08-17 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Moraxella catarrhalis exempte d'endotoxine
WO2006085910A3 (fr) * 2004-06-04 2006-09-28 H And Human Services The Gover Moraxella catarrhalis exempte d'endotoxine

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AU2003200964B2 (en) 2004-11-11
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