KR20010034124A - Lipooligosaccharide-based vaccine for prevention of Moraxella(Branhamella) catarrhalis infections in mammals - Google Patents

Abstract

Isolated from Moraxella (Branhamella) catarhalis (hereinafter referred to as M. catarhalis) to remove ester bound fatty acids to remove non-toxic lipooligosaccharides (dLOS) or lipid A. A conjugate vaccine comprising lipooligosaccharide non-toxicized with an immunological carrier to which a non-toxic oligosaccharide (OS) is constructed. Vaccines are useful for preventing otitis media and respiratory infections caused by M. catarhalis in mammals, including humans.

Description

Lipooligosaccharide-based vaccine for prevention of Moraxella (Branhamella) catarrhalis infections in mammals}

M. catarrhalis is the third most frequent cause of otitis media and sinusitis in children, following Streptococcus pneumoniae and Haemophilus influenzae (H. influenzae). Known pathogens (Bluestone, CD, Dr μg 31 (Suppl 3): 132-41, 1986; Catlin, BW, Clin.Microbiol. Rev. 3: 293-320, 1990; Doern, GV, Diagn. Microbiol.Infect.Dis. 4: 191-201, 1986; Enright, MC & H. Me. Kenzie, J. Med. Microbiol. 46: 360-71, 1997; Faden, H. et al., J. Infect. 169: 1312-1317, 1994). Such Gram-negative diplococci are also found in adults (Boyle, FM et al., Med. J. Aust. 154: 592-596, 1991; Sarrubi, FA et al., Am. J. Med. 88: 9S-14S). , 1990), particularly in adults with immunocompromised or chronic lung disease (Enright, MC & H. MeKenzie, J. Med. Microbiol. 46: 360-371, 1997). The incidence by M. catarrhalis is increasing (see McLeod, DT et al., Br. Med. J. 292: 1103-1105, 1986; Fung, CP et al., J. Antimicorb. Chemother. 30 : 47-55, 1992). To date, no vaccine against M. catarrhalis-borne diseases is known.

Antigens immunizing against M. catarrhalis are not clear, but the development of serum antibodies against M. catarrhalis is important in terms of immunity against M. catarrhalis. For example, normal adults who are naturally retained or immune due to infection have a lower retention rate (1 to 6%) than children (50 to 78%) and older people (26% or more) Less frequently (see Ejlertsen T. et al. J. Infect. 29: 23-31, 1994; Faden H. et al., J. Infect. Dis. 169: 1312-1317, 1994, Eliasson, I., Dr μg 31 (Suppl 3): 7-10, 1986; Vaneechoutte, M. et al., J. Clin.Microbiol. 28: 2674-2680, 1990). Children gradually form serum antibodies against M. catarrhalis during the four years of life, which may be associated with reduced incidence of bacteremia and otitis media caused by M. catarrhalis (see CDR Weekly Reports, Communicable Disease Surveillance Centre, London). , 1992-1995; Goldblatt D. et al., J. Infect. Dis. 162: 1128-1135, 1990; Vaneechoutte, M. et al., J. Clin.Microbiol. 28: 2674-2680, 1990; Bluestone, CD, Dr μg 31 (Suppl 3): 132-141, 1986). Antibodies against M. catarrhalis are also found in the serum of acute or recovering adult patients (see Christensen, KJ et al., Clin. Diagn. Lab. Immunol. 3: 717-721, 1990; Rahman, M. et al. , APMIS 105: 213-220, 1997). Serum from most recovering patients shows bactericidal activity against M. catarrhalis isolates (Chapman, A.J. Jr. et al., J. Infect. Dis. 151: 878-882, 1985). These results suggest that serum antibodies are associated with the prevention of M. catarrhalis infection.

To date, research on M. catarrhalis as an important pathogen has generally focused on describing surface antigens such as outer membrane proteins (OMPs) (Bhushan, R. et al., J. Bacteriol. 176: 6636-6643). , 1994; Campagnari, AA et al., Infect. Immun. 62: 4909-4914, 1994; Helminen, ME et al., Infect. Immun. 61: 2003-2010, 1993; Helminen, ME et al., J. Infect. Dis. 170: 867-872, 1994; Murphy, TF et al., Mol. Microbiol. 10: 87-97, 1993). UspA, a high molecular weight protein, and CD protein, a major outer membrane protein, are the most widely studied outer membrane proteins. These proteins are relatively conserved against different M. catarrhalis species and can form bactericidal antibodies (Helminen, ME et al., J. Infect. Dis. 170: 867-872, 1994; Murphy, TF et al., Mol. Micorobiol. 10: 87-97, 1993; Yang, YP et al., FEMS Immunol. Med. Micorobiol. 17: 187-199, 1997). In addition, when mice were immunized manually with monoclonal antibodies against UspA or immunized with UspA, lung pulmonary clearance to M. catarrhalis was increased (Helminen, ME et al. J. Infect. Dis. 170: 867-872, 1994; Chen, D. et al., Infect. Immun. 64: 1900-1905, 1996). The genes encoding the CD protein were cloned and their base sequences analyzed (Murphy et al., Molec. Microbiol. 10 (1): 87, 1993).

Other outer membrane proteins that were purified and characterized were protein E (OMP E) (Bhushan et al., J. Bacteriol., 176 (21): 6636, 1994), protein B1 (Ducey et al. , Abstracts, Gen. Mtg. Am. Soc. Micobiol. 96 (0): 186, 1996) and protein COPB (Aebi et al., 1996, Abstracts, Intersci.Conf.Antimicrobial Agents & Chemotherapy 36: 158, 1996). Include. Other surface antigens include cilia and polysaccharides, which are not found in all isolates (Marrs, CF & S. Weir, Am. J. Med. 88 (suppl 5A): 36S-40S, 1990). The presence or absence of capsular polysaccharides is controversial (Ahmed, K. et al., Micorobiol. Immunol. 35: 361-366, 1991). High molecular weight envelope proteins associated with lipooligosaccharides have also been identified (Klingman & Murphy, Abstr. Gen. Mtg. Am. Soc. Micobiol., 1992).

Lipooligosaccharide (LOS: lipooligosacchride, hereinafter referred to as 'LOS') is a major surface element of and is a virulence agent that causes onset of bacterial infections (Doyle, WJ, Pediatri. Infec. Dis. J. 81 (suppl): S45 -S47, 1989; Fomsgaard, JS et al., Infect. Immun. 59: 3346- 3349, 1991). LOS was found in (1) M. catarrhalis-infected patients, and (2) anti-LOS immunoglobulin G (imgoglobulin G, hereinafter referred to as 'IgG') in convalescent patients. Activity, and (3) because LOS appears to have a conserved structure according to serological properties in humans (Rahman, M. et al., Eur. J. Clin Micorobiol. Infec. Dis. 14: 297 -304, 1995; Tanaka, H. et al., J. Jpn. Assoc. Infec. Dis. 66: 709-715, 1992). Similarly, to other microorganisms (eg, H. influenza type b, Neisseria meningitidis, Vibrio cholerae, Shigella sonnei). Antibodies that specifically have a bactericidal activity in serum, such as lipopolysaccharides (hereinafter referred to as 'LPS') or polysaccharides (hereinafter referred to as 'PS'), confer immunity against these pathogens in humans. Robbins, JB et al., J. Infec. Dis. 171: 1387-1398, 1995; Cohen D. et al., Lancet 349: 155-159, 1997).

Three major antigenic types of LOS (A, B and C) account for 95% of M. catarrhalis (ie, 61% A; 29% B; 5% C in one study) (Vaneechoutte, M.). et al., J. Clin. Micorobiol. 28: 182-187, 1990). Studies have shown that these LOSs have oligosaccharides linked to lipid A without O-specific polysaccharides, and these three antigenic types have one inner core in common and diverge to their respective antigenic types (Edebrink, P). et al., Carbohydr.Res. 257: 269-284, 1994; Edebrink, P. et al., Carbohydr.Res. 266: 237-261, 1995; Edebrink, P. et al., Carbohydr.Res. 295 : 127-146, 1996).

LPS and LOS derived from various microorganisms are largely toxic in mammalian in vivo. Many approaches have been attempted to detoxify LPS or LOS, or to obtain non-toxic polysaccharides or oligosaccharides from LPS or LOS, respectively. For example, a weak acid treatment of LPS or LOS was used as a method of separating lipid A from LOS molecules in the vicinity of Kdo-glucosamine linkage (see Gu, XX, & CM Tsai, Infect. Immun. 61: 1873-1880, 1990). Another method is the weak alkali treatment of LOS, which preserves the amide-linked fatty acids of lipid A while removing ester-linked fatty acids (see Gupta, RK et al., Infect. Immun. 60). : 3201-3208, 1992; Gu et al., Infect. & Imm. 64 (10): 4047, 1996).

A variety of methods have been used to develop vaccines against M. catarrhalis and other microorganisms (Karma et al., Int'l. J. Ped. Othorhinolaryngol. 32 (suppl.): S127-S134, 1995). Outer membrane proteins E and CD, derived peptides, oligopeptides and nucleic acids encoding these proteins are disclosed in US Pat. Nos. 5,607,846 and 5,556,755. A conjugate vaccine made by combining a carbohydrate-containing antigen with a cytokine, lymphokine, hormone or growth factor with immunomodulatory function is disclosed in US Pat. No. 5,334,379. Canadian Patent No. 2,162,193 discloses the use of purely purified lactoferrin receptor as a vaccine against pathogens that produce lactoferrin receptor proteins, including M. catarrhalis.

WO 90/11777 discloses a method for obtaining bacterial cilia subunits which are not assembled for use in vaccines against M. catarrhalis and other bacteria.

In mammals, particularly in children and adults, development of a vaccine that is non-toxic and immune to M. catarrhalis is needed to prevent otitis media, sinusitis or similar respiratory infections. Although methods for detoxifying LOS in other microorganisms are known, these non-toxicized products (ie heptenes) are generally less immune in vivo. Therefore, there is a need for a form of M. catarrhalis LOS that is nontoxic but capable of producing an immune response that is preferably sufficient to produce anti-LOS antibodies, such as IgG, in mammalian in vivo.

Summary of the Invention

In one aspect of the invention it is isolated from M. catarrhalis to remove the esterified fatty acid to obtain detoxified lipooligosacharide (hereinafter referred to as 'dLOS'), or lipid A to remove only oligosaccharides (OS) A conjugate vaccine against M. catarrhalis constructed by covalently binding a lipooligosaccharide (LOS) obtained by detoxification by one method to an immunogenic carrier is disclosed. In one embodiment the immune carrier is a protein, and in another embodiment the protein is UspA, CD, tetanus toxin / denaturing toxin isolated from M. catarrhalis, Haemophilus influenzae (hereinafter, High molecular weight protein (HMP) isolated from H. influenzae), diphtheria toxin / modified toxin, non-toxic P. aerµginosa toxin A, cholera toxin / modified toxin, pertussis toxin / modified toxin, CRM ₁₉₇ as an exotoxin / denatured toxin of Clostridium perfringens, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 protein (see Pappenheimer et al. Immunochem. 9: 891-906) , 1972) and CRM ₃₂₀₁ (Black et al., Science 240: 656-659, 1998) and cross reacting materials (CRM) and respiratory syncytial virus F and G proteins. In one aspect of the vaccine the immune carrier protein is tetanus denatured toxin or high molecular weight protein. In another embodiment, there is a pharmaceutical composition comprising such a conjugate vaccine in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition may comprise an adjuvant. Preferably the adjuvant used here is a mixture of monophosphate lipid A with trehalose dimycolic acid or alum. In one embodiment the immune carrier is covalently bound to the deesterified LOS via a linker compound. Preferably, the linking compound is selected from adipic acid dihydrazide, ε-aminohexane, chlorohexanol dimethyl acetal, D-glucuronolactone, p-nitrophenyl amine, and more preferred linking compound is adipic acid dihaye It's a drazide. In one embodiment the vaccine further comprises covalently binding the isolated oligosaccharide (OS) to one immunocarrier by removing lipid A from the LOS of M. catarrhalis.

Another aspect of the present invention discloses a non-toxic lipooligosaccharide (dLOS) and an oligosaccharide obtained by removing lipid A from LOS by isolating LOS from M. catarrhalis and removing ester-bound fatty acids from LOS. In one embodiment, M. catarrhalis from which LOS is isolated is a purely isolated species.

Another aspect of the present invention discloses a method for preventing otitis media due to M. catarrhalis infection in a mammal, which is obtained by removing lipid A from dLOS or LOS which detoxified LOS derived from M. catarrhalis. A conjugate vaccine constructed by covalently binding an OS to an immunological carrier comprises administering to the mammal an amount effective in immunological terms. In one preferred embodiment the mammal is a human and in another embodiment the conjugate vaccine is administered by intramuscular injection, subcutaneous injection or deposition into the nasal mucosa, or a combination of the foregoing methods. In another embodiment, the immunologically effective dose is 10-50 μg one time and the method is inoculated again after 13 months of administration of the 10-25 μg conjugate vaccine at about 2 months. It also includes a method. In one embodiment, about 10 months after the first dose of 10-25 μg of the conjugate vaccine, about 2 months after the second dose of about 10-25 μg of the conjugate vaccine, then about 12 months After this time, the administration step of the fourth inoculation of 10 to 25 μg of the conjugate vaccine.

In another aspect of the invention, a method of detoxifying LOS isolated from M. catarrhalis is disclosed, including a method for removing ester bound fatty acids from LOS. In one embodiment the ester bound fatty acid can be removed by hydrazine or weak alkali treatment.

The present invention also includes a method of making LOS by removing lipid A from the LOS as a method for detoxifying LOS, and in one embodiment, lipid A is removed by acid treatment.

In another aspect of the present invention, a method for constructing a conjugate vaccine against M. catarrhalis is disclosed. The ester-linked fatty acid is removed from LOS isolated from M. catarrhalis to obtain a deesterified LOS (dLOS). A method of covalently binding to an immunological carrier. In one embodiment, the removing step comprises treatment of hydrazine or weak alkali to the LOS. In one embodiment, the binding step comprises binding the dLOS to a linker compound and again binding the linking compound to an immunological carrier. If preferred, the linking compound is adipic acid dihydrazide, ε-aminonucleic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, p-nitrophenyl amine and more preferred linking compound is adipic acid dihydrazide . In one embodiment the adjuvant may be included in the vaccine composition.

The present invention aims to prevent otitis media caused by M. catarrhalis infection in mammals, to remove non-toxic esterified fatty acids or lipid A to remove oligosaccharides from M. catarrhalis to obtain non-toxic LOS (dLOS). Lipo-oligosaccharide (LOS) obtained by detoxifying by obtaining only (OS) is covalently coupled to one immunological carrier (Immunogenic carrier) to provide a conjugate vaccine against M. catarrhalis. If desired, the immune carrier is a protein, and in one preferred embodiment, the immune carrier is from UspA, CD, tetanus toxin / denatured toxin isolated from M. catarrhalis, Haemophilus influenzae of type indeterminate Isolated high molecular weight protein (HMP), diphtheria toxin / modified toxin, non-toxic P. aeruginosa toxin A, cholera toxin / modified toxin, pertussis toxin / modified toxin, Clostridium perfringens ) Retavirus VP 7 protein or Respiratory Syndrome Virus F and G proteins, such as exotoxins / modified toxins, hepatitis B surface antigen, hepatitis B core antigen, cross reacting materials (CRM) including CRM ₁₉₇ and CRM ₃₂₀₁ . Preferably, the immune carrier protein is tetanus toxoid (hereinafter referred to as 'TT') or high molecular protein (hereinafter referred to as 'HMP').

The present invention relates to a conjugate vaccine for preventing bacterial infection. More specifically, the present invention is a vaccine against human Moraxella (Branhamella) catarrhalis (hereinafter, referred to as 'M. catarrhalis') infection, which is obtained from lipooligosaccharide isolated from the bacterium. The present invention relates to a vaccine in which an esterified fatty acid or lipid A is removed and an immunogenic carrier is bound.

1 is a graph showing the bactericidal titration of M. catarrhalis (species 25238) of anti-M. Catarrhalis serum obtained from 2-3 rabbits, in which each rabbit was LOS ("LOS"), conjugate ("dLOS-TT). "And" dLOS-HMP "), or conjugates (" dLOS-TT + Ribi "and" dLOS-HMP + Ribi ") were vaccinated twice with the adjuvant. Bactericidal titration is expressed as a fold increase in serum values prior to inoculation, based on serum dilution ratios that can kill bacteria at least 50% for each group, expressed as geometric mean (bar) and standard deviation (line above bar). It was. The sterilization titration ratio of hyperimmune sera to whole cells of M. catarrhalis was 1: 1600.

FIG. 2 is a diagram showing a passive immune protection study plan using pulmonary clearance of M. catarrhalis (species 25238) in a rat against haze. Forty mice were inoculated with rabbit anti-dLOS-TT serum or pre-vaccination rabbit serum and then challenged with M. catarrhalis in the haze for 18 hours. Samples were taken from rats aged 3 and 6 hours after challenge.

FIG. 3 is a graph showing the results of the passive immune protection study described in FIG. 2. Lung and blood samples were taken for analysis, and the y-axis shows bacterial colony forming units (CFU) per lung. The first bar shows the control group and the second group received the vaccine. Three hours after challenge, the amount of bacteria in the vaccine group was reduced by 50% compared to the control. Six hours after challenge, there was a 61% reduction in the vaccine group compared to the control group.

LOS from Moraxella (Branhamella) catarhalis is a major surface antigen that induces sterile antibodies against bacteria that cause otitis media and sinusitis in children and in adults respiratory infections. For the sake of simplicity, hereinafter, the electric bacteria will be called Moraxella catarhalis or M. cartarhalis. The LOS of M. cartarhalis was isolated and processed to reduce its toxicity by approximately 20,000 times when analyzed using the LAL test. Non-toxic LOS (dLOS) can determine the type of carrier (eg tetanus toxin / denaturing toxin (TT) or type) using a linker compound to form dLOS-TT or dLOS-HMP. To pure purified high molecular weight protein (HMP) from H. influenzae. The molar ratio of dLOS to TT and HMP when constructing the conjugate was 19: 1 and 31: 1, respectively. The antigenicity of these two conjugates was similar to that of the isolated LOS when subjected to double immunodiffusion analysis. In addition, when these two conjugates were inoculated subcutaneously (s.c.) or intramuscularly (i.m.), the average level of IgG against LOS was increased. In mice, after three inoculations of the conjugate, the average IgG level increased by 50 to 100-fold, and in rabbits, the IgG level was increased by 350-700-fold after 2 inoculations. Immunity of the conjugate can be further enhanced by the addition of an adjuvant when constructing the conjugate.

In rabbits, antiserum produced by vaccination of conjugates induced complement-mediated bactericidal activity against both single pure and heterogeneous mixed species of M. cartarhalis. These results show that non-toxic LOS-protein conjugates are useful as vaccines for diseases caused by M. cartarhalis.

Purely isolated type A M. Cartarhalis (ATCC strain 25238, available from the US Center for Reference Cultures (Rockville, MD)) is a standard method (Edenbrink, P. et al., Carbohaydr. Res. 257: 269-). 284, 1994; Masoud, H. et al., Can. J. Chem. 72: 1466-1477, 1994) as one exemplary strain capable of isolating LOS. For other known M. cartarhalis species, in many cases these are either clinical isolates that can be obtained from ATCC or other storage sites, or are purely isolated by well-known bacteriological methods, and the use of them as strains of LOS is also present invention. It is contained within the realm of. Briefly, M. cartarhalis species 25238 was incubated in chocolate agar medium for 8 hours, then inoculated in 3% tryptic soybean broth (TSB) and incubated overnight at 37 ° C. The culture is diluted and transferred to a flask with a stopper containing TSB and incubated for another 24 hours at 37 ° C. The cells are precipitated by centrifugation, and the precipitated cells are washed with ethanol, acetone and petroleum ether using standard methods and then dried as a powder (Masoud, H. et al., Can. J.). Chem. 72: 1466-1477, 1994). LOS containing less than 1% protein and nucleic acid (Smith, PK et al., Anal. Biochem. 150: 76-85, 1985; Warburg, O. & W. Christian, Biochem. Z. 310: 384- 421, 1942) to modify the standard method using phenol and water (Westphal, O. et al., Methods Carbohydr. Chem. 5: 83-91, 1965) (Gu, XX., Infect). Immu. 63: 4115-4120) to extract the LOS, and another known method of isolating LOS may replace the former method.

Although hydrazine is used in the present invention to detoxify LOS isolated from M. cartarhalis, lipids, such as a method of treating weak alkalis using diluted (0.1N) NaOH or other diluted base solutions with a pH of 13.2 to 13.6, The use of other enzymes or reagents capable of removing ester-bound fatty acids from A is also included in the scope of the present invention. It is important to use a detoxification method that is gentle enough so that the oligosaccharide portion of the LOS is not hydrolyzed. Hydrolysis of oligosaccharides destroys the epitope that provides immunoprotection. Isolated M. Cartarhalis was previously described (Gu, XX., Infect. Immu. 64: 4047-4053, 1996; Gupta, RK et al., Infect. Immu. 60: 3201-3208, 1992 ), Nontoxic through anhydrous hydrazine treatment under fairly light conditions. Briefly, LOS is suspended in anhydrous hydrazine and incubated at a temperature of 1-100 ° C., preferably at 25-75 ° C., more preferably at about 37 ° C. Incubate with mixing for 10 minutes to 24 hours, preferably about 2 to 3 hours, with the addition of cold acetone until precipitates are formed that allow the mixture to cool and centrifuge. The precipitate is washed with acetone and then dissolved in water and ultracentrifuged. Subsequently, the supernatant is taken, lyophilized, and then dissolved again, followed by column chromatography to obtain fractions containing carbohydrates. The fractions are collected, lyophilized and named dLOS. By weight, dLOS is approximately 38% of LOS.

Alternatively, as described by Gu et al., LOS can be detoxicated by mild lipid treatment with diluted or weak acid at pH 2-3 to obtain oligosaccharides (OS) by removing lipid A. (Infect. Immu. 61: 1873-1880, 1993). This OS is bound to the carrier in the same way as in dLOS. Although the present invention refers to the use of acetic acid to remove lipid A from LOS of M. cartarhalis, the use of other reagents or enzymes capable of removing lipid A is also within the scope of the present invention. OS-protein conjugates elicit immune responses in both rats and rabbits, and induce antibodies against both LOS and carrier proteins. In rats, sera immunized with conjugates (with adjuvants) showed bactericidal activity against M. cartarhalis mono pure species 25238. Rabbits also exhibit bactericidal activity against homologous strain 25238 in serum immunized with conjugates.

To prepare dLOS or LOS conjugates, dLOS can be directly bound to the carrier protein using a crosslinking reagent such as, for example, glutaraldehyde. Preferably, dLOS or LOS conjugates are constructed using linker compounds through a variety of methods (see Marburg et al., J. Am. Chem. Soc. 108: 5282, 1986; US Pat. No. 4,882,317). Nos. 5,153,312 and 5,204,098), linking compounds separate each other between dLOS or OS and a carrier protein, promote the effective linkage of dLOS or OS to a carrier and optimize the immunity of the conjugate. The linking compound, whose length and flexibility can be adjusted as desired, can distinguish the carbohydrate moiety from the carrier element and increase the translational and rotational properties of the conjugate antigen, thereby increasing the access of the antibody. The linking compound between the antigen and the carrier has a variety of structural properties, including heteroatoms and cleavage sites. Adipic acid dihydrazide is the preferred linking compound, but there are other suitable linking compounds such as, for example, ε-aminonucleic acid, chloronuxanol dimethyl acetal, D-glucurunolactone, p-nitrophenyl amine. Linking reagents contemplated for use in the present invention include hydroxysuccinimide and carbodiimide. Many suitable linking compounds and linking reagents are known to those skilled in the art (Dick et al., Conjugates Vaccines, JM Crues & RE Lewis, Jr., eds., Karger, New York, pp. 48-114, 1989). .

In one preferred embodiment, the dLOS or OS is first derivatized with adipic acid dihydrazide (ADH), which functions as a linking compound to the protein carrier. Briefly, in order to form adipic acid dihydrazide (ADH) to form adipic dihydrazide-dLOS (AH-dLOS) or adipic dihydrazide-OS (AH-OS), a known method is used to form a Kdo residue carboxyl group of dLOS or LOS. (Gu, XX, & CM Tsai, Infect. Immun. 61: 1873-1880, 1993). The excess molar ratio of ADH was used to prevent dLOS-dLOS linkage and for effective conjugate linkage. The molar ratio of ADH to dLOS or OS in the reaction mixture is typically about 10: 1 to 250: 1, preferably about 50: 1 to 150: 1, more preferably about 100: 1. In one preferred embodiment, there is one ADH per dLOS or OS in the AH-dLOS conjugate. In another embodiment, in the final dLOS-carrier conjugate, the molar ratio of dLOS or OS to the carrier is about 15 to 100, with the preferred lower range being about 20 to 35 and the preferred upper range. Is about 40 to 75, with a preferred range of about 25 to 50, and more preferably about 50. This ratio can generally be adjusted while varying the initial starting concentration and reaction time of AH-dLOS or AH-OS and the carrier. In general, there is a positive correlation between the proportions in this range and the antibody response to the conjugate in vivo (ie, the higher the ratio, the higher the immune response). Column chromatography is performed to obtain a fraction containing both carbohydrate and adipic acid dihydrazide from the reaction mixture, to obtain derivatized dLOS and OS (Kemp, AH & MRA Morgan, J. Immunol. Methods 94: 65-72, 1986). The former fraction is collected, lyophilized and named AH-dLOS or AH-OS.

In a preferred embodiment of the present invention, a variety of carriers known in the art have been combined with dLOS or OS of M. cartarhalis to one protein, more preferably tetanus toxin (TT) or high molecular weight protein (HMP) isolated from H. Influenzae. Are suitable for forming the dLOS- or OS-carrier conjugates of the invention. HMP is a major antigen of antibodies in the serum of convalescent patients infected with H. Influenzae, belonging to a group of high molecular weight proteins exposed on the cell surface (see Barenkamp for more structural and functional characterization. J. Infect. Dis 165 (Suppl. 1): S181-184, 1992). The carrier increases the immunity of the oligosaccharide and the antibodies formed against the carrier may be medically useful. The carrier may be water soluble or insoluble, and may be a material comprising, for example, primary and / or secondary amino groups, azido groups, carboxyl groups as natural or artificial polymerization immunological carriers. Any of a variety of immunological carrier proteins can be used to construct the dLOS- or OS-conjugates of the invention. Such proteins include, for example, cilia, outer membrane proteins of pathogens, secretory toxins, nontoxic or "denatured" forms of such secretion toxins, antigens to bacterial toxins (known as cross-reactants or CRMs) and other proteins. It may contain similar, but non-toxic, proteins. Preferred outer membrane proteins are those isolated from Gram-negative bacteria, including UspA and CD isolated from the outer membrane of M. cartarhalis, denatured toxins are also preferred. Examples of bacterial toxins and denatured toxins contemplated for use in the present invention include, but are not limited to, tetanus toxins / modified toxins, high molecular weight proteins isolated from H. influenzae of type indeterminate, diphtheria toxins / modified Toxin, non-toxic P. aeruginosa toxin A, cholera toxin / denatured toxin, pertussis toxin / denatured toxin, Clostridium perfringens exotoxin / denatured toxin. The use of viral proteins (eg, hepatitis B surface antigen or core antigen; Rotavirus VP 7 protein and Respiratory Syndrome Virus (RSV) F and G proteins) as carriers has also been contemplated.

Cross reacting material (CRM) includes CRM ₁₉₇ , which is antigenically equivalent to diphtheria toxin, and CRM ₃₂₀₁ , a variant genetically engineered pertussis toxin. For example, the use of immunological carriers derived from non-mammalian animals such as keyhole limpet hemocyanin, horseshoe crab hemocyanin, and plant estestin is also present. It is included within the scope of the invention.

In forming dLOS- or OS-protein conjugates of M. cartarhalis, several linkage methods have been envisioned. For example, as presented herein, dLOS or OS is derivatized with AH and bound to TT or HMP. Another way to generate suitable dLOS- or OS-protein conjugates is to destabilize dLOS with cystamine, which is N-succimidyl-3- (2-pyridyldithio) after EDC-mediated derivatization. Disulfide conjgation to a propionate-derivatized protein (N-succimidyl-3- (2-pyridyldithio) propionate-derived protein). Other well known methods of binding oligosaccharides to other immunological carrier proteins are also described in the scope of the invention, as described, for example, in US Pat. Nos. 5,153,312 and 5,204,098 and in EP 0 497 525 and EP 0 245 045. Included.

AH-dLOS or AH-OS is coupled to tetanus toxin (TT) or high molecular weight protein (HMP) of H. influenzae to construct the conjugate (Gu, XX, & CM Tsai, Infect. Immun. 61: 1873). -1880, 1993). The molar ratio of AH-dLOS or AH-OS relative to protein elements in the reaction mixture is typically about 10: 1 to 250: 1, preferably about 50: 1 to 150: 1, more preferably about 100: 1 to be. For example, AH-dLOS is dissolved in water and mixed with these proteins in a molar ratio of about 100: 1 relative to TT or HMP. Then, the pH was adjusted to 5.4 ± 0.2, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrogen chloride (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide HCl) was added to the reaction mixture. The reaction was stirred for 1 to 2 hours. The reaction mixture was adjusted to pH 7.0 again, centrifuged and purified by column chromatography. Fractions containing both carbohydrates and proteins were recovered and named dLOS-TT or dLOS-HMP, depending on the protein used to construct the conjugate. The composition of carbohydrates and proteins in the conjugates was analyzed by conventional methods using dLOS and BSA as standard materials (Dubois, M. et al., Anal. Biochem. 28: 250-256, 1956; Smith, PK et al., Anal. Biochem. 150: 76-85, 1956). Those skilled in the art will appreciate whether the dLOS or OS linked to the carrier is from a single M. cartarhalis species or from several species to be active against several strains. Alternatively, the dLOS- or OS-carrier conjugate can be constructed using dLOS or OS separately for the construction of a single conjugate, followed by production of a vaccine comprising one or more dLOS- or OS-carrier conjugates. Different binders may be used in order to mix them. In this way, a vaccine comprising one or more of the known antigenic types for M. cartarhalis LOS can be constructed.

Pure purified dLOS was characterized and compared with pure purified LOS using standard SDS-polyacrylamide electrophoresis (SDS-PAGE) and silver staining methods, as previously described. (Tsai, CM & CE Frasch, Anal. Biochem. 119: 115-119, 1982). Preparative M. cartarhalis LOS (25, 50, 100, 200ng) and dLOS (20ug) were electrophoresed together using LPS Ra and Rc from Salmonella minnesota as standard materials. In the lanes containing 20 μg of dLOS, protein bands were hardly visible at the LOS position, whereas each lane containing M. cartarhalis LOS showed a single band with a molecular weight of about 4,000 (see Edenbrink, P. et al. , Carbohydr.Res. 257: 269-284, 1973). In lanes containing 20 μg of dLOS, faintly spread bands were observed at the position of the S. minnesota LPS Ra band instead. These results show that dLOS contains less than 0.25% residual M. cartarhalis LOS.

Toxicity of isolated LOS and dLOS was tested by standard Limulus (LAL) assays (Hochstein, H.D., et al, Bull.Paperer.Drg Assoc. 27: 139-148, 1973). Using standard materials available from the US FDA, the sensitivity of the LAL assay is 0.2EU / μg. dLOS showed 1EU / μg, while isolated LOS showed 20,000EU / μg with a 20,000-fold reduction in toxicity. Preferably, a composition showing a reduction of about 500 to 1000 fold or more in terms of EU / μg may be used as a vaccine. For example, in vitro toxicity reductions determined using LAL assays are associated with significantly acceptable levels of in vivo toxicity reduction. Biotoxicity can be readily determined using standard biopyrogen test methods (eg, 0.1-1 μg / kg body weight in rabbits).

The antigenicity of the dLOS, AH-dLOS, dLOS-TT and dLOS-HMP conjugates was tested using double immunodiffusion with hyperimmune serum on whole M. cartarhalis (species 252238) cells. . High immunity serum was prepared using standard methods. Briefly, two New Zealand rabbits (female, 2-3 kg per horse) were injected subcutaneously and intramuscularly twice (inoculated with both sc and im methods per inoculation) at 10 ⁹ M. cartarhalis (species 252238) at 4 week intervals. Inoculate a suspension (1: 1 volume ratio) of a mixture of whole cells and incomplete Freund's adjuvant. Blood samples were taken before each inoculation and 2 weeks later.

High immunity sera produced a sharp settling band that could easily be observed in response to LOS in a dual immunodiffusion assay. Similarly, high-immune serum also reacted with dLOS to form a rather widespread sedimentation band, indicating that the isolated dLOS retained the antigenicity of the isolated LOS. The high immune serum also reacted with the dLOS-TT and dLOS-HMP conjugates, forming the same precipitation bands when compared to LOS. In contrast, highly immune serum did not react to measurably with isolated HMP.

Antigenicity can be measured using an enzyme-linked immunosorbent assay (ELISA) by modifying known methods (Gu, X.X. et al., Infect. Immun. 64: 4047-4053, 1996). ELISA plates were coated with LOS and blocked with 3% BSA. ELISA wells were reacted with diluted rabbit serum before the addition of alkaline phosphatase bound goat's anti-rabbit IgG and IgM (Sigma). Between all procedures, the wells were washed with a large amount of phosphate buffered saline (PBS) containing a polymerization inhibitor (0.01% Tween-20). The substrate of the enzyme was added for 30 minutes and the absorbance (405 nm) of the reaction was measured. The antigenicity of the dLOS-carrier conjugate was determined in a similar manner using the conjugate as the coating antigen and the immunoserum of the diluted rabbit as the binding antibody. Both types of dLOS-carrier conjugates showed similar binding to rabbit high immune serum, and the antigenicity of dLOS-carrier conjugates was higher than that of LOS conjugates under the same conditions.

To measure in vivo toxicity, dLOS-carrier conjugates were inoculated parenterally into rats and rabbits, and the levels of serum anti-LOS antibodies in each animal were measured using ELISA. Mixtures of unbound dLOS with TT or HMP did not elicit anti-LOS antibodies. In contrast, each combination of dLOS-TT and dLOS-HMP induced low levels of anti-LOS IgG after inoculation of the conjugate. After tertiary inoculation of the conjugate, anti-LOS IgG levels increased 50-100 fold. dLOS-TT and dLOS-HMP both induced similar levels of anti-LOS IgG after three inoculations. Inoculation with LOS alone or with dLOS-carrier conjugate induced similar levels of anti-LOS IgG.

The combination of dLOS-TT and dLOS-HMP conjugates with adjuvant enhanced immunity in rats. In other words, two inoculations of the dLOS-carrier conjugate with an adjuvant resulted in similar or higher levels of IgG than three inoculations of the conjugate alone. Three inoculations of the dLOS-carrier conjugate with the adjuvant showed a 9-15 fold increase in anti-LOS IgG levels obtained after three inoculations of the same conjugate without the adjuvant. When inoculated with the composition of the conjugate-adjuvant three times, dLOS-TT induced lower levels of IgG than dLOS-HMP.

Adjuvants used contained monophosphate Lipid A and trehalose dimycolic acid (available as a product under the name Ribi-700 from Ribi Immunochemical Research, Hamilton, MT). For example, aluminum compounds (ie, alum), chemically modified lipooligosaccharides, suspensions of sterile Pertussis pertussis, N-acetylmural-L-alanyl-D-glutamine, other adjuvants The use of other standard adjuvants commonly known in the art as well is within the scope of the present invention. Additional adjuvants have been described by Warren et al. (Ann. Rev. Biochem. 4: 369-388, 1986; New Generation Vaccines, 2nd Edition, Levine, MM et al., Eds., Marcel Dekker, Inc., NY, 1997). The use of aluminum compounds as the adjuvant is preferred, and the use of adjuvant licensed to humans is particularly preferred. When rat serum was analyzed at IgM levels, LOS was inoculated tertiary and showed high levels of IgM of anti-LOS, whereas the conjugate induced low or medium anti-LOS antibodies at each inoculation. The addition of adjuvant to the dLOS-protein conjugate improved the anti-LOS IgM levels produced after the second inoculation.

When the mouse serum was analyzed in the same way for the protein elements (TT or HMP) of the dLOS-protein conjugate, antibodies to these proteins were found. With respect to anti-TT antibodies, dLOS-TT induced low levels of IgG at the first inoculation, which levels increased significantly after the second and third inoculations. Inoculation of the dLOS-TT conjugate with adjuvant induced increased IgG levels than mice inoculated with the same conjugate without adjuvant. The combination of TT and dLOS induced higher levels of IgG than the anti-TT IgG induced by dLOS-TT. All immunogens induced low levels of anti-TT IgM, which was further increased with the use of adjuvant when inoculated.

When the serum of mice was analyzed in the same way for anti-HMP antibodies, dLOS-HMP induced low levels of anti-HMP IgG after the first inoculation, and the anti-HMP IgG significantly increased after the second and third inoculations. Improved. Administration of the adjuvant in mice given the dLOS-HMP conjugate increases anti-HMP IgG levels. Inoculation with simply mixing HMP and dLOS resulted in anti-HMP IgG levels higher than the anti-HMP IgG levels found in mice inoculated with or without dLOS-HMP (adjuvant Table 2). All immunogens induced low levels of anti-HMP IgM.

Immunogenicity of the dLOS-protein conjugate was determined immediately after subcutaneous and intramuscular injection in rabbits and about a month later (inoculation in two ways, subcutaneous and intramuscularly per inoculation). Blood samples were taken at 0 hours (ie at the time of the first inoculation), two weeks after the first inoculation, and then at the second week. In order to determine the antigenicity of rat serum, the levels of IgM and IgG were measured for the serum of rabbits inoculated with the following immunogens (50 μg per inoculation): LOS, dLOS-TT, DLOS-TT, dLOS-HMP with adjuvant, dLOS-HMP with adjuvant, and a simple mixture or non-binding mixture of three substances (dLOS, TT and HMP) were inoculated with M. cartarrhalis cells.

Administration of a simple mixture of dLOS, TT and HMP or LOS alone showed low levels of anti-LOS IgG or IgM after two inoculations. The dLOS-TT conjugate induced high levels of anti-LOS IgG after primary and secondary inoculation compared to serum before inoculation. Inoculation of dLOS-TT conjugates induced lower levels of IgG than inoculation of dLOS-HMP. Inclusion of adjuvant at each inoculation increased anti-LOS IgG in both conjugates, and when inoculated twice with adjuvant, there was no significant difference in anti-LOS IgG levels of the two conjugates. For IgM, these two conjugates induced low or moderate levels of anti-LOS antibodies, with adjuvant combined resulting in higher anti-LOS IgM than did not normally use.

When the rabbit serum was analyzed in the same way for antibodies produced against the protein elements (TT or HMP) of the dLOS-protein conjugate, antibodies to that protein were found. For anti-TT antibodies, dLOS-TT induced low levels of IgG at the first inoculation, but the level increased significantly after the second inoculation. The combination of TT, dLOS, and HMP showed higher levels of anti-TT IgG than the inoculation of dLOS-TT, compared with the use of adjuvant. After inoculation, the levels were lower than the anti-TT IgG produced. All immunogens induced low or moderate levels of anti-TT IgM.

For anti-HMP antibodies, dLOS-HMP induced low levels of IgG at the first inoculation, but the level increased significantly after the second inoculation. When inoculated with dLOS-HMP with adjuvant, further increased anti-TT IgG was observed. Inoculation with TT, dLOS and HMP showed higher levels of anti-HMP IgG than inoculation with dLOS-HMP, but after inoculation of dLOS-HMP with adjuvant, It was rather low. All immunogens induced low or moderate levels of anti-HMP IgM.

Antisera produced in rats and rabbits were tested for in vitro bactericidal activity against pure single and heterogeneous M. cartarhalis using standard methods (Gu, XX et al., Infect. Immun. 64: 4047-4053, 1996). In rabbits, serum immunized with LOS or dLOS had no bactericidal activity against pure single species M. cartarhalis. In contrast, sera immunized with dLOS-TT showed bactericidal activity at an average ratio of 1:16 (without adjuvant) and 1:40 (including adjuvant) and 1:10 for dLOS-HMP, respectively. Bactericidal activity was observed at the mean titration ratio of 1:40 (without adjuvant). According to the ELISA assay, anti-LOS IgG levels were correlated with the observed bactericidal titration ratios.

When the antisera activity of the antiserum produced against pure species of M. cartarhalis and mixed species in other regions was measured, antiserum against dLOS-protein conjugate in rabbits was higher than that produced in the same way. The antiserum of all rabbits induced by the conjugate showed bactericidal activity against pure single species M. cartarhalis, and the representative antiserum showed activity against most non-pure species (10 ATCC species and 9 of clinical isolates). Indicated. In contrast, less than half of the antiserum of rats induced by the same method showed bactericidal activity against pure species. In general, there is a correlation between bactericidal titration and the level of anti-LOS IgG antibodies.

The bactericidal activity of the antiserum of all rabbits induced by dLOS-TT with adjuvant was additionally analyzed using 20 M. cartarhalis. Ten of the 20 species were complement or serum sensitive strains. When the serum of the rabbits was analyzed using the remaining 10 species, the bactericidal activity of the four ATCC strains and 9 clinical isolates was 1:15 (in the range of 1: 2 to 1:32). One species showed negative results in bactericidal activity assay.

In rats, 20% (4 out of 20 mice) of seroimmunized with dLOS-carrier conjugates and 45% (9 out of 20 mice) of sera immunized with adjuvant (9 out of 20 mice) Low sterilization titration rates were obtained for M. cartarhalis (ATCC 25238).

The results presented in the examples of the present invention show that after detoxification, M. cartarhalis dLOS retains antigenic determinants, but by itself is not immune in vivo. When dLOS is linked to a protein carrier, the dLOS element is immune. That is, the dLOS-protein carrier of M. cartarhalis induces significant levels of IgG antibodies against LOS in mammals. In mammals dLOS-carrier conjugates induce anti-LOS IgG antibodies to levels similar to LOS. Immunogenicity of dLOS-protein conjugates is better in rabbits than rats (ie, multiples of anti-LOS antibodies when vaccinated twice in rabbits with the same conjugate are shown twice after inoculation into rats two or three times Generally higher than the increase). In both animals, the level of anti-LOS antibody was increased when the conjugate was inoculated with the adjuvant rather than without the adjuvant. Both dLOS-TT and dLOS-HMP conjugates induced similar levels of anti-LOS antibodies, which increased when the conjugate was formulated as an adjuvant.

The dLOS-protein carrier of M. cartarhalis of the present invention is useful as a vaccine capable of inducing immunity to M. cartarhalis infection in preventing otitis media and respiratory diseases in mammals, especially in humans. The dLOS-protein carrier construction method disclosed herein may be usefully used in constructing such vaccines. The methods disclosed herein are also useful for identifying other dLOS-protein carriers (ie, dLOS conjugates linked to other carrier residues) capable of inducing an immune response against M. cartarhalis in mammals, particularly in humans, including children. Do. Vaccines against M. cartarhalis include dLOS-carrier conjugates with immunologically inactive but pharmaceutically acceptable reagents or clinically acceptable adjuvants. One skilled in the art may target the dLOS-protein carrier of M. cartarhalis with immunologically active elements against other infectious agents (eg, M. cartarhalis and one or more other bacteria or viruses that cause disease in children). Combination vaccine). For example, there are triple vaccines targeting M. cartarhalis, H. influenzae and S. pneumoniae, whose type cannot be determined, to prevent otitis media.

For vaccination, the dLOS-carrier conjugate can be administered parenterally. Several methods of vaccination have been considered, including methods of muscle (im), subcutaneous (sc), abdominal (ip), mucosal (eg, via nasal mucosa), and arterial vaccinations. desirable. For parenteral administration, the dLOS-carrier conjugate may be prepared sterile, for example, sterile water-soluble or oily inoculum with or without adjuvant. Such injections may be prepared using reagents used for dispersing, soaking or suspending, according to various methods well known to those skilled in the art. Sterile fluid preparations can be parenterally acceptable diluents or solvents, such as sterile injectable solutions or 1,3-butaneniol. Other suitable diluents can include, for example, water, Ringer's solution, isotonic saline. In addition, sterile, fixed oils may be conventionally employed as a solvent or suspending medium. For example, fixed oils in which nothing is added, including synthetic monoglycerides or diglycerides, can be used. In addition, fatty acids such as oleic acid may be used to prepare the injection solution. As for the nasal mucosal administration method, the carrier may be prepared using an inert carrier (eg, air or hydrocarbon) and various conventional methods. It may be administered by a mist method.

In the vaccine of the present invention, the dLOS-carrier conjugate may be water-soluble or microparticulate, and may be in a microsphere or microvesicle including liposomes. In one embodiment, the amount of conjugate administered can be about 10-100 μg, preferably about 20-50 μg. In another preferred embodiment, the dosage is about 25-40 μg. The exact dosage can be determined according to routine dosage / response protocols that are well known to those skilled in the art, ie generally as seen in children, in particular, for determining the dosage per body weight.

The vaccine of the invention can be administered to mammals of all ages and can be adapted to induce active immunity against otitis media, respiratory infections caused by M. cartarrhalis in young mammals, especially humans. As a child vaccine, the dLOS-carrier protein may be administered about 2 to 12 months of age, and preferably between about 2 to 6 months. A booster vaccination may be given and typically between about 10 and 25 μg booster may be given, for example, about 2 months and 13 months after the first inoculation. Alternatively, booster vaccination can occur about 4 months and 16 months after primary vaccination, and other booster vaccinations were also considered.

The composition of the vaccine involves the use of a combination of derivatives from different M. cartarrhalis species to protect against all or most medically relevant species. Three types of M. cartarrhalis are known based on dLOS: Types A, B and C account for 61%, 29% and 5% of clinical isolates, respectively. As shown in Example 6, the antiserum for one species, if not all, can cross react with several other species. Therefore, different binders may be mixed and used as a vaccine. Mixed combinations containing dLOS or OS isolated from Forms A, B, and C can react with 95% of all medically relevant species, while containing dLOS or OS isolated from Forms A and B When combined, they can react with 90% of all medically related species.

As described in Example 7 of the present invention, passive immunoprotection studies were conducted in which mice were immunized with rabbit antiserum against dLOS-TT and challenged with M. cartarrhalis sp. 25238 in the room. In the group administered the vaccine, the number of bacterial CFUs per lung was significantly reduced. These rat lung clearance models mimic the natural spread of bacteria in humans. The advantages of this model are that it is simple, repeatable, well tuned by nebulizers, large numbers of mice can be studied under the same opposing conditions, and no surgical procedure is required for bacterial inoculation.

While antimicrobial antibodies against dLOS-carrier conjugates are not expected to be associated with specific modes of action or mechanisms, antimicrobial antibodies, particularly IgG, may be released to the nasopharyngeal mucosa surface. Antibodies on mucosal surfaces can inactivate M. cartarrhalis inoculated on mucosal surfaces, thereby preventing or alleviating symptoms of otitis media and respiratory disease caused by M. cartarrhalis. Secretory IgA may play an important role in respiratory mucosal immunity if the conjugate vaccine is administered to the nasal mucosa.

The following examples illustrate some preferred embodiments of the invention.

Example 1: Pure Purification and Nontoxicization of LOS from M. cartarrhalis

M. cartarrhalis (type A) ATCC 25238 was used as an exemplary strain for pure purification of LOS (Edebrink, P. et al., Carbohydr. Res. 257: 269-284, 1994; Masoud, H. et. al., Can. J. Chem. 72: 1466-1477, 1994). The strain was incubated for 8 hours using a chocolate agar medium at 37 ℃, 5% carbon dioxide conditions, 250ml of 3% tryptic soybean broth medium (TSB, Difco Laboratories, Detroit, Mich.) Transferred to the medium. The culture bottles were incubated overnight at 37 ° C., 110 rpm shaker (Model G-25, New Brunswick Scientific, Co., Edison, NJ). The culture was transferred to a 2.8 L Fernbach flask with 6 valve plates each containing 1.4 L of TSB, and the temperature was maintained at 37 ° C. for 24 hours while shaking at 110 rpm. The culture was centrifuged at 15,000 × g for 10 minutes at 4 ° C. to recover cells.

Precipitated cells were washed once with 95% ethanol, twice with acetone and twice with petroleum ether using conventional methods and then dried to a powder (Masoud, H. et al., Can. J. Chem. 72: 1466-1477, 1994). Modification of standard methods using phenol and water (Westphal, O. et al., Methods Carbohydr. Chem. 5: 83-91, 1965) (Gu, XX, Infect. Immun. 63: 4115-4120 , 1995) to extract LOS from cells, which can produce LOS containing less than 1% protein and nucleic acid (Smith, PK et al., Anal. Biochem. 150: 76-85, 1985; Warburg, O. & W. Christian, Biochem. Z., 310: 384-421, 1942.

LOS was treated with anhydrous hydrazine as a weak alkaline treatment condition to remove ester bound fatty acids from Lipid A (Gu, XX et al., Infect. Immun. 64: 4047-4053, 1995; Gupta, RK et al. , Infect. Immun. 60: 3201-3208, 1992). Briefly, LOS (160 mg) was suspended in 16 mL of anhydrous hydrazine (Sigma Chemical Co., St. Louis, Mo.) and incubated with mixing at 37 ° C. for 3 hours. This suspension was cooled on ice and added with cold acetone dropping until a precipitate formed. The mixture was centrifuged at 5,000 xg for 30 minutes at 5 ° C, and the precipitate was washed twice with cold acetone, dissolved in pyrogen-free water at a concentration of 10 to 20 mg per mL, 150,000 rpm for 3 hours at 5 ° C. Ultracentrifugation. Subsequently, the supernatant was lyophilized and passed through a Sephadex G-50 column (1.6 cm in diameter, 90 cm in length, Pharmacia LKB Biotechnology, Uppsala, Sweden), eluted with 25 mM ammonium acetate and subjected to a differential refractometer (R-400; Waters, Milford, Mass). The eluate was analyzed for carbohydrates using a fine phenol-sulfuric acid method (see Dubois, M. et al., Anal. Biochem. 28: 250-256, 1956). Fractions containing carbohydrates were recovered, lyophilized and named dLOS, corresponding to about 38% of LOS.

Non-toxicizing M. cartarrhalis LOS in this manner is a method of removing lipid A from the Kdo-glucosamine linkage by mild acid treatment of LOS (Gu, XX, & CM Tsai, Infect. Immun. 61: 1873-1880). , 1993), after linking to a protein carrier, which yields higher yields.

Example 2: Derivatization of dLOS and Binding with Proteins

The adipic hydrazide (hereinafter referred to as 'AH') derivative of dLOS prepared by the method of Example 1 was constructed and purified as follows. Adipic acid dihydrazide using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrogen chloride (EDC) and N-hydrosulfide succinimide (sulfo-NHS) (Pierce): Hereinafter, 'ADH', Aldrich Chemical Co., Milwaukee, Wis) was coupled to the carboxyl group of the Kdo residue of dLOS to generate an AH-dLOS derivative (see Gu, XX, & CM Tsai, Infect. Immun. 61). 1873-1880, 1993). Briefly dLOS (70 mg) was dissolved in 7 mL of 345 mM ADH (based on the expected molecular weight of dLOS (3,000), the molar ratio of ADH to LOS is about 100: 1) (Edebrink, P. et. al., Carbohydr.Res. 257: 269-284, 1994). Then sulfo-NHS was added at a concentration of about 8 mM, the pH was adjusted to 7.0, and then EDC was added at a concentration of 0.1 M. The reaction mixture was stirred and maintained at pH 4.8 for 3 hours. The reaction mixture was adjusted to pH 7.0, passed through a G-50 column as described in Example 1, and the eluate was analyzed for carbohydrates and adipine hydrazide (AH) (see Kemp. AH & MRA Morgan). , J. Immunol.Methods 94: 65-72, 1986). Fractions containing both carbohydrates and AH were recovered, lyophilized and named AH-dLOS. AH-dLOS analyzed the composition using dLOS and ADH as standard materials.

AH-dLOS was linked to protein (TT or HMP) in the following manner. TT (Connaµght Labs, Inc., Swiftwater, Pa.) And HMP were purely isolated from Haemophilus influenzae species 12, whose type could not be determined (see Barenkamp SJ). , Infect. Immune., 64: 1246-1251, 1996). AH-dLOS was linked to TT or HMP to generate a conjugate (Gu, X.X., & C.M. Tsai, Infect. Immun. 61: 1873-1880, 1993). Briefly, AH-dLOS (30 mg) was dissolved in 3 mL of water and mixed with 15 mg of TT (5.9 mg / ml) or 12 mg of HMP (4 m / mL). The molar ratio of AH-dLOS to TT (molecular weight 150K) and HMP (molecular weight 120K) was about 100: 1. The pH was adjusted to 5.4 and EDC was added at a concentration of about 0.05 to 0.1 M. The reaction mixture was then maintained at pH 5.6 for 1-3 hours with stirring. The reaction mixture was adjusted to pH 7.0 and centrifuged to pass through a Sephacryl S-300 column (1.6 cm in diameter, 90 cm in length) containing 0.9% NaCl. Fractions containing both protein and carbohydrate were recovered and named dLOS-TT or dLOS-HMP, respectively. These two conjugates were analyzed for carbohydrate and protein composition using dLOS and ADH as standard materials (Duboi, M. et al., Anal. Biochem. 28: 250-256, 1956; Smith, PK, et al. , Anal.Biochem. 150: 76-85, 1985).

Derivatized AH-dLOS and dLOS-protein conjugates were characterized by physical properties based on the measurement of the content of AH and dLOS (μg / ml) or dLOS and protein in the conjugate. Based on the carbohydrate content, the yield of AH-dLOS was about 93%. For dLOS-TT, 103 μg of dLOS and 266 μg of protein were detected per ml; As for dLOS-HMP, 220 μg of dLOS and 280 μg of protein were detected per ml. The molar ratio of the conjugate was calculated as the number of moles of dLOS per mole of protein using a molecular weight of 3,000 for dLOS, 150,000 for TT, and 12,000 for HMP. The yield of the binder was calculated using the phenol-sulfuric acid method based on the initial starting amount of dLOS and the amount of dLOS contained in the binder. Yield was 8% for dLOS-TT and 19% for dLOS-HMP.

Example 3: Antigenicity of dLOS, Derivatized dLOS and dLOS-Protein Conjugates

The antigenicity of dLOS, AH-dLOS and conjugate was analyzed by double immunodiffusion and enzyme-linked immunosorbent assay (ELISA) using the serum of hyperimmune rabbits against whole cells of M. cartarrhalis (species 25238). . High immunity sera were prepared as described previously, and dual immunodiffusion was performed by standard methods using 0.8% agar gel in phosphate buffered saline (PBS, pH 7.4). In this experiment the central wells contained serum highly immunized with M. cartarrhalis whole cells, and the surrounding wells each included: 1 mg LOS per ml, 103 μg dLOS-TT, 220 μg dLOS-HMP (based on the amount of dLOS), 1 mg and 200 μg dLOS and 500 μg HMP. In dual immunodiffusion assays, highly immune sera produced a sharp settling band that could easily be observed in response to LOS. Similarly, highly immune serum also reacted with dLOS (at two concentrations tested) to form a rather widespread sedimentation band, indicating that the isolated dLOS retained the antigenicity of the isolated LOS. The high immune serum also reacted with the dLOS-TT and dLOS-HMP conjugates to form a clear precipitation band. These two conjugates and LOS formed a fairly identical precipitation band in the double immunodiffusion assay. In contrast, highly immune serum did not react to measurably with isolated HMP.

ELISA was performed by modifying the previously described method as follows (Gu, X. X. et al., Infect. Immun. 64: 4047-4053, 1996). The wells of the standard ELISA edition (Dynatech Laboratories, Inc., Alexandria, VA) were coated with LOS, then blocked with 3% BSA for 1 hour in PBS and for 2 hours by adding rabbit serum (1 / 8,000 dilution) Incubated. Then, anti-rabbit IgG and IgM (Sigma) of goats bound with alkaline phosphatase were added and incubated for 1 hour. The wells were washed with PBS containing polymerization inhibitor (0.01% Tween-20) between all procedures. After the enzyme substrate was added for 30 minutes, the reaction was measured for absorbance at 405 nm using a Microplate autoreader (EL309, Bio-Tek Instruments). The antigenicity of the dLOS-carrier conjugate was determined as ELISA activity by absorbance measurement at 405 nm in the same manner. The conjugate was determined as the coating antigen (10 μg / ml) and the immune serum of the diluted rabbit was used as the binding antibody (1 / 8,000 dilution).

For the two conjugates described in Example 3, both responded similarly to the serum of highly immunized rabbits. The absorbance value (A ₄₀₅ ) of dLOS-TT was 1.9 and dLOS-HMP was 1.3. Under the same conditions, LOS (10 μg / ml) showed a value of 1.1 (A ₄₀₅ ).

In vivo immunogenicity of the dLOS-protein conjugate was investigated in the following animal models: rats and rabbits.

Example 4: Immunogenicity of dLOS-protein Conjugates in Mice

Female rats 10 to 20 weeks of age per group were given 5 μg (based on carbohydrates) of dLOS-TT, dLOS-HMP, LOS, alone, or dLOS and TT or HMP (5 μg of protein) per inoculation, respectively. A mixture of the two proteins was added to 0.9% NaCl and injected subcutaneously with or without adjuvant. The adjuvant is 50 μg of monophosphate lipid A (MPL) and 50 μg of synthetic trehalose dimycolic acid on an inert carrier (available under the trade name Ribi-700 from Ribi ImmunoChem Research, Ltd. (Hamilton, Mt.)). (STD). Inoculations were performed three times at three week intervals, and on day 14 of the first inoculation, blood samples were taken from rats 7 days after the second and third inoculations.

The level of anti-LOS serum was expressed in ELISA units (hereinafter referred to as 'EU') using LOS isolated from 25238 species as coating antigen. As standard sera, high immunity sera for whole species 25238 were used and assigned a value of 65,000 EU for IgG and 800 EU for IgM per ml. Serum antibodies against TT or HMP were also analyzed using ELISA, where 5 μg of TT or HMP per ml was used as the coating antigen, and after three inoculations of TT or HMP, ELISA was performed based on the serum of the resulting mice. Expressed in units (EU). Rat sera used as standard sera had a value of 2,000EU for IgG per ml and 10EU for IgM.

To statistically analyze these results, the level of antibody was expressed as the geometric mean of the ELISA unit (EU) or n independent observations ± standard deviation or inverse titer of the category (n <4). Significance was verified by a two-sided t test and P values less than 0.05 were considered significant.

As shown in the data in Table 1, simply mixing dLOS with TT or HMP did not induce anti-LOS antibodies. Two conjugate vaccines, dLOS-TT and dLOS-HMP, did not induce anti-LOS antibodies at the first inoculation, but induced low levels of anti-LOS antibodies after the second inoculation. After tertiary inoculation, anti-LOS antibody levels increased by 50-100 fold (p <0.01). Both inoculations of dLOS-TT and dLOS-HMP induced similar levels of anti-LOS IgG after three inoculations. LOS alone induced similar levels of anti-LOS IgG as with conjugates.

The combination of DLOS-TT and dLOS-HMP with Ribi adjuvant increased the immunogenicity of the conjugate. Two inoculations of the adjuvant and the conjugate were similar to three inoculations with the conjugate alone, or induced high levels of IgG, and three inoculations induced a 9-15 fold increase in anti-LOS IgG ( p <0.05). The dLOS-TT conjugate induced lower levels of IgG when formulated with the adjuvant than when inoculated three times with dLOS-TT.

For IgM, LOS induced high levels of anti-LOS IgM after the third inoculation, while the conjugate induced low or medium levels of anti-LOS IgM after each inoculation. Ribi adjuvant increased anti-LOS IgM levels in the conjugate group.

Rat antibody response to M. catarrhalis LOS induced by the conjugate Immunogen ^a Blood sample ^b Geometric mean (± standard deviation range) ^c ELISA units IgG IgM dLOS + TT 123 113 11 (1-2) 5 (1-15) dLOS + HMP 123 111 (1-2) 11 (1-2) 1 dLOS-TT 123 15 (1-19) 52 (6-447) ^** 17 (2-23) 17 (3-91) dLOS-HMP 123 12 (1-8) ^* 101 (15-691) ^** 1 (1-2) 6 (2-19) 17 (5-63) dLOS-TT + Adjuvant 123 3 (1-11) * 210 (48-923) ^** 470 (266-828) 2 (1-4) 16 (62-396) 14 (8-25) dLOS-HMP + Adjuvant 123 3 (1-9) ^* 101 (26-389) ^** 1,514 (299-7,658) 9 (2-39) 22 (6-84) 32 (13-80) LOS 123 18 (2-40) ^* 113 (20-630) ^** 3 (1-9) 2 (1-4) 52 (12-230)

a: 5 μg of LOS three times at three week intervals of 10-20 rats in each group; 5 µg

concrete; 5 μg of conjugate containing Ribi adjuvant; LOS; And, dLOS and TT

Or a mixture of HMP (5 μg each) was injected subcutaneously.

b: Blood samples were taken as follows. 1, 2 weeks after the first inoculation;

2, 1 week after the second dose; 3 and 3 weeks after 1 week

c: ELISA unit based on reference serum for strain 25238

LOS obtained from strain 25238 was coated with a coating antigen.

Used. The data marked with (*) and (**) of a single immunogen show the

There was a significant difference (p <0.01).

In the data shown in Table 2, dLOS-TT showed low levels of anti-TT IgG antibody after one inoculation, and markedly increased after two and three inoculations (p <0.01). Inoculation of Ribi adjuvant enhanced the level of IgG in the dLOS-TT group, and the mixture of TT and dLOS expressed higher levels of IgG than that formed by dLOS-TT. All immunogens expressed low levels of anti-TT IgM.

In addition, Table 2 shows the results for the anti-HMP antibody. The dLOS-HMP conjugate expressed low levels of IgG after one inoculation and increased significantly (p <0.01) after two and three inoculations. Ribi adjuvant enhanced the level of IgG in the dLOS-HMP group, and the mixture of HMP and dLOS expressed higher levels of IgG than dLOS-HMP. All immunogens were expressed at low levels of anti-HMP IgM.

Mouse antibody response to a protein (TT or HMP) induced by the conjugate Immunogen ^a Blood sample ^b Geometric mean (± standard deviation range) ELISA units IgG IgM TT dLOS-TT 123 1 (1-2) 34 (21-54) 90 (35-237) 112 (1-3) dLOS-TT + Adjuvant 123 14 (6-35) 303 (191-481) 2,430 311 (7-18) 27 (13-56) dLOS + TT 123 11 (7-18) 303 (191-481) 729 11 (1-2) 2 (1-3) Anti-HMP dLOS-HMP 123 12 (1-8) 11 (3-43) 111 dLOS-HMP + Adjuvant 123 4 (2-9) 52 (30-92) 810 (389-1,685) 2 (1-6) 7 (3-17) 11 (7-18) dLOS + HMP 123 2 (1-6) 377 (149-952) 1,403 (645-3,051) 2 (1-3) 2 (1-3) 10 (7-14)

a: 5 μg of LOS three times at three week intervals of 10-20 rats in each group;

5 μg of conjugate mixed with Ribi adjuvant; And dLOS and TT or HMP (respectively

5 μg) was injected subcutaneously.

b: Blood samples were taken as follows: 1, 2 weeks after the first inoculation;

2, 1 week after the second inoculation; 3 and 3 weeks after 1 week

Example 5: Immunogenicity of dLOS Protein Conjugates in Rabbits

LOS, dLOS-TT conjugate without adjuvant, dLOS-TT conjugate without adjuvant, dLOS-HMP conjugate with adjuvant, adjuvant 50 μg of each inoculum was administered to the dLOS-HMP conjugate without addition and simply a mixture of TT or HMP in dLOS. If no adjuvant was added, the immunogen was dissolved in 1 ml of 0.9% NaCl. When adjuvant was added, the immunogen was dissolved in 1 ml of 0.9% NaCl containing 250 μg of single phosphate lipid A and 250 μg of trihalose dimycolic acid (Ribi-700 adjuvant, Ribi Immunochemical Research, Hamilton, MT). I was. Two weeks after the inoculation, 10-20 ml of blood was collected from the rabbit's ear vein by standard methods.

In Table 3, the level of anti-LOS IgG or IgM was low after two inoculations with a mixture of dLOS, TT and HMP or LOS alone. dLOS-TT conjugates significantly increased the levels of anti LOS IgG after 1 and 2 inoculations (37 and 700 fold, respectively, compared to preimmune serum). dLOS-HMP showed lower levels of IgG than dLOS-TT (six times and 347 times respectively compared to preimmune), and Ribi adjuvant enhanced anti-LOS IgG levels in both conjugate groups after inoculation, respectively. (40-2000 fold increase compared to preimmune). There was no major difference between the reactions of the two conjugates after two doses.

In the case of IgM, both conjugates had lower than average levels of anti-LOS antibodies, and those with Ribi adjuvant also showed lower levels of anti-LOS antibodies after inoculation.

Response of Rabbit Antibodies to Moraxella catarrhalis LOS Obtained by Conjugates Immunogen ^a Blood sample ^b Geometric mean (± standard deviation range) ELISA units IgG IgM LOS 012 6 (3-10) 10 (3-30) 52 (30-90) 6 (3-10) 52 (30-90) 90 dLOS-TT 012 5 (3-10) 187 (90-270) 3,505 (2,430-7,290) 14 (10-30) 90187 (90-187) dLOS-TT + Adjuvant 012 10810 (270-2,430) 21,870 5 (3-10) 389 (270-810) 270 (90-810) dLOS-HMP 012 7 (3-10) 43 (30-90) 2,430 1030 (10-90) 90 (30-270) dLOS-HMP + Adjuvant 012 10 (3-30) 389 (30-2,430) 21,870 7 (3-10) 90 (30-270) 270 (90-810) dLOS + TT + HMP 012 10 (3-30) 10 (3-30) 52 (30-90) 17 (10-30) 3030 Whole cells 012 6 (3-10) 27065,610 10156 (90-270) 49 (3-810)

a: 2-3 rabbits in each group subcutaneously and intramuscularly in 0 hours and 1 month

50 μg LOS; 50 μg of conjugate; A conjugate mixed with 50 μg Ribi adjuvant; In addition

Was immunized with 50 μg each of a mixture of dLOS, TT and HMP. M.

Highly immunized rabbit serum for catarrhalis is described in the present invention.

b: Blood samples were taken as follows. 0: before immunization

1: 14 days after first dose 2: 2: 14 days after second dose

The data for anti-TT antibodies shown in Table 4 showed that dLOS-TT showed a significant increase in IgG after two inoculations (more than 389 times compared to pre-immune serum). Ribi adjuvant appeared to be four times more elevated in levels of IgG than by dLOS-TT after two inoculations. The mixture of TT and dLOS specifically showed higher levels of anti-TT IgG than dLOS-TT conjugates after a single inoculation. All immunogens induced low levels of anti-TT IgM.

In Table 4, for anti-HMP antibodies, dLOS-HMP significantly increased IgG levels after two inoculations (81-fold higher than pre-immune serum). After Ribi adjuvant was added, after two inoculations The level of IgG is four times better than that of dLOS-HMP. The mixture of HMP and dLOS specifically showed higher levels of IgG than dLOS-HMP after a single inoculation. All immunogens induced low levels of anti-HMP IgM.

Rabbit antibody response to proteins (TT or HMP) represented by dLOS-protein conjugates Immunogen ^a Blood sample ^b Geometric mean (± standard deviation range) ^b ELISA units IgG IgM TT dLOS-TT 012 37 (3-10) 1,168 (810-2,430) 31014 (10-30) dLOS-TT + Adjuvant 012 5 (3-10) 14 (10-30) 5,055 (2,430-21,870) 314 (10-30) 10 (3-30) dLOS + TT + HMP 012 102702,430 109030 Anti-HMP dLOS-HMP 012 10 (3-30) 14 (10-30) 810 7 (3-10) 1010 dLOS-HMP + Adjuvant 012 7 (3-10) 130 (30-270) 3,505 (2,430-7,290) 10 (3-30) 14 (10-30) 30 dLOS + TT + HMP 012 10156 (90-270) 2,430 103052 (30-90)

a: 2 to 3 rabbits in each group subcutaneously and intramuscularly in 0 hours and 1 month

At 50 μg LOS; 50 μg of conjugate; 50 μg binding combined with Ribi adjuvant

sieve; Each mixture of dLOS, TT and HMP was immunized with 50 μg. M.

Highly immunized rabbit serum for catarrhalis is described in the present invention.

b: Blood samples were taken as follows. 0: before immunization

1: 1 day after 1st inoculation 2: 2: 1 day after 2nd inoculation

Example 6: Bactericidal Activity of Animal Serum against M. catarrhalis Strains

In this example, the bactericidal activity of the animal serum prepared according to Examples 4 and 5 was compared with the strain from which LOS was isolated (ATCC 25238: "pure single strain") and other wild strains of M. catarrhalis ("heterologous strain"). Was carried out. Eleven M. catarrhalis wild strains (ATCC No. 8176, 8193, 23246, 25238, 25239, 25240, 43617, 43618, 43627, 43628, 49143, purchased from ATCC, Rockville, MD) and from patients with otitis media and respiratory infections The clinically isolated strain (named M1 to M10) was tested. The practitioner will find that additional strains of M. catarrhalis were easily isolated from clinical specimens using standard bacteriological methods and tested in a similar manner.

For bactericidal analysis, the serum of rabbits before and after immunization (after two inoculations) was incubated at 56 ° C. for 30 minutes to inactivate the complement components. The inactivated serum was then measured for bactericidal activity against M. catarrhalis using a known method of complement mediated bactericidal assay (Gu X. X. et al., Infect. Immun. 64: 4047-4053, 1996). The difference was that guinea pig serum was used as a complement source (1: 1 dilution, 20 μl per well), and the reaction medium was incubated for 30 minutes at 37 ° C. prior to loading into agar medium. The highest dilution factor of serum showing 50% or more killing effect was shown as back sterilization titration.

When mice were used, 20% of conjugate immune serum (4 of 20 rats) or 45% of conjugate immune serum with adjuvant (9 of 20 rats) after three inoculations with dLOS-TT or dLOS-HMP ) Showed low titer bactericidal effect against pure single strains.

In the graph of Figure 1, the serum of animals immunized with LOS (or dLOS) in rabbits did not show bactericidal activity against pure single species M. catarrhalis (ATCC 25238). In contrast, sera immunized with dLOS-TT showed bactericidal activity with an average titration of 1:16 when adjuvant was absent and 1:40 when adjuvant was present. Serum immunized with dLOS-HMP showed no adjuvant. The mean titration was 1:10 if none and 1:40 when adjuvant was added. There was a correlation between r = 0.60 and p = 0.02 between LOS IgG ELISA level and bactericidal titration, but there was no correlation between IgM levels and bactericidal titration.

The bactericidal activity of rabbit antiserum exhibited by dLOS-TT with Ribi adjuvant was further analyzed with 10 additional wild strains (ATCC strains) and 10 strains clinically isolated from Japan. Ten of the 20 strains were complement sensitive (strains 23246, 43617, M9) or serum sensitive (strains 43627, 43628, 49143, M4, M7, M8, M10). As a result of experiments using the remaining 10 strains, rabbit antiserum showed bactericidal activity with an average ratio of 1:15 (1: 2 to 1:32) against four ATCC strains and five Japanese strains. One strain (ATCC 25240) had no bactericidal activity. Cross-reactivity seen in bactericidal activity showed that vaccines capable of generating an immunoprotective response against all virulence strains of M. catarrhalis could be formed from a relatively small number of conjugates constructed from dLOS from different species. .

Example 7: Passive Immunoprotective Study in Lung Clearance Model of Rats

Forty mice were immunized with rabbit antiserum, or pre-immune sera against dLOS-TT, and then challenged for 18 hours with 10 ⁸ CFU of M. catarrhalis species 25238 in the nebula. After 3 and 6 hours of fighting, rat lung and blood samples were taken for analysis (see FIG. 2), and the results are shown in FIG. 3. After 3 hours, the number of bacteria in the vaccine group was reduced by 50% compared to the control group. After 6 hours of challenge, the number of bacteria in the vaccine group was reduced by 61% compared to the control group. There was a significant difference between vaccine groups. Antibody levels were analyzed, which was inversely related to the colony forming units of the bacteria. These results indicated that antibodies induced by dLOS-TT conjugates improved lung clearance of M. catarrhalis in rats.

Example 8: Human Immunization with M. catarrhalis dLOS-Protein Conjugate

Adults were initially selected to test the stability and immunogenicity of the dLOS-carrier conjugate prepared as shown in Examples 1 and 2 or the OS-carrier conjugate prepared as shown in Examples 9-11. Screening tests were performed to identify individuals with relatively low levels of endogenous antibodies (eg, those who had childhood infection with M. catarrhalis) and to study adults with relatively low levels of antibodies compared to the general population. Selected for the purpose. The selected individuals were then inoculated with dLOS-TT conjugate, dLOS-HMP conjugate, OS-TT conjugate or OS-HMP conjugate using a pharmaceutically acceptable carrier (25-50 μg depending on body weight). In adults, a single inoculation is usually sufficient to trigger an antibody response within three to two weeks. The immunogenicity and bactericidal activity of the resulting antiserum were determined using the method described in Examples 4-6. The second inoculation was performed approximately 1 to 6 months after the first inoculation, and the level of anti M. catarrhalis antibody in serum was measured after about one week. The control group was inoculated with the control vaccine prepared in the same amount of the protein portion of each conjugate using a pharmaceutically acceptable carrier and the inoculation plan was the same as the electroimmunization method.

In individuals who received dLOS or OS-carrier conjugates, serum antibody levels after immunization indicated that the conjugates were immunogenic without incurring any adverse effects in vivo, and bactericidal activity was associated with serum antibodies of these individuals. have. In some individuals, multiple vaccinations were desirable to elicit appropriate antibody responses. In contrast, sera obtained from the control group receiving control inoculation did not show measurable immunogenicity or anti-M. Catarrhalis bactericidal activity compared to that seen in serum obtained prior to inoculation. This means that antiserum obtained from adults inoculated with dLOS-TT conjugate, dLOS-HMP conjugate, OS-TT conjugate or OS-HMP conjugate showed more potent immunogenic or anti-M. Catarrhalis bactericidal activity than the control group. . In addition, the incidence of otitis media infection in each individual was examined over the years, with no one of the adults immunized with dLOS-TT, dLOS-HMP, OS-TT or OS-HMP conjugates during the period.

Based on the anti-serum results obtained from adults, the vaccine was extended to children aged 2 to 4 months. The vaccine was first inoculated with the vaccine (10 to 40 µg depending on weight), followed by 2 months and 4 months. At the end of the month, two boosters were given. One group of children received three boosters over the 2, 4 and 16 months after the first inoculation, and children of similar age who did not receive the inoculation were used as controls. Serum antibodies of immunized children were examined and immunogenicity and bactericidal activity were measured as before. After the first inoculation, otitis media and sinusitis were observed in children up to 4 years of age. The vaccinated children had a significantly reduced incidence of otitis media and venous sinusitis. Decreased.

Example 9: Oligosaccharide (OS) Formation by Acid Hydrolysis of M. catarrhalis Strain 25238 LOS

M. catarrhalis LOS removes lipid A at the Kdo-glucosamine linkage site of LOS by mild acid treatment of LOS (ie, Gu, XX and CM Tsai method, Infect. Immun. 61: 1873-1880, 1993), Oligosaccharides form and become nontoxic. In summary, 421 mg of LOS was dissolved in distilled water to make 10 mg / ml, and then hydrolyzed with 1% acetic acid at 100 ° C. for 2-3 hours. The lipid A portion and unhydrolyzed LOS were removed by centrifugation at 150,000 × g for 3 hours. The supernatant was lyophilized, dissolved in a small amount of water, separated into two and each placed in a Sephadex G-25 column (1.6 × 90 cm) balanced with 25 mM ammonium acetic acid. The eluate was analyzed to determine the sugar content, and the major OS fractions were collected and lyophilized three times to remove salts. The amount of OS obtained from the start of LOS is 47% by weight.

Example 10 Induction of OS by ADH

ADH is linked to the OS by carbodiimide mediated condensation reactions using EDC and sulfo-NHS, as described previously (Gu et al., 1993, supra.). The reaction mixture was separated by a Bio-Gel P-4 column (1.6 × 90 cm; Bio-Rad, Hercules, CA) to obtain an eluate. The eluate was analyzed for sugar and AH content, and sugar and AH content. All high fractions were pooled and lyophilized three times to remove salts. The ratio of OS to ADH was approximately 1 and the yield of sugar in the AH-OS derivative was about 74%.

Example 11: Binding of AH-OS to Proteins

The ligation reaction was carried out at pH 5.2 ± 0.2 using 0.05-0.1 M EDC. For the OS-TT reaction, 30 mg of AH-OS was dissolved in 3 ml of distilled water and mixed with 15 mg of TT (5.90 mg / ml), 20 mg of AH-OS was dissolved in 2 ml of water for the OS-HMP reaction. Mix with 8.4 mg (4.2 mg / ml) HMP. The molar ratio of TT or HMP to AH-OS is 150 or 141: 1 (2,000 for OS; 150,000 for TT; by molecular weight 120,000 for HMP). The separation step is the same as for the dLOS protein conjugate described in Example 2. Such OS-protein conjugates have the same utility as dLOS-protein conjugates as vaccines against Moraxella catarrhalis infection in humans, as described above. Composition, yield and antigenicity for the OS-TT and OS-HMP conjugates are shown in Table 5.

Composition, yield and antigenicity of OS conjugates concrete Amount (μg / ml): ^A molar ratio for OS / protein Yield ^b (%) A ₄₀₅ ^c (high immunological serum) OS protein OS-TT1OS-TT2OS-HMP1OS-HMP2 23434732 157152182112 11211617 2.46.18.84.5 1.10.62.01.4

a: the ratio is 2,000 for molecular weight dLOS, respectively; 120,000 for TT; HMP

It is expressed as the moles of OS against the moles of protein that is 120,000.

b: The amount of OS contained in the starting amount of OS and the conjugate is determined by the fine phenol sulfate method.

Measured.

c: The antigenicity of the conjugate is that the conjugate is used as a coating antigen (10 µg / ml)

Absorbance at A ₄₀₅ when high immune serum was used as binding antibody (1 / 8,000)

It is shown by ELISA activity. For LOS (10 μg / ml), A ₄₀₅ under identical conditions

Absorbance at represents 1.1.

OS-protein conjugates showed antibody responses in both rats and rabbits to LOS. The protein conjugate also produced antibodies against TT and HMP in rats and rabbits. Mouse antibody responses to M. catarrhalis strain 25238 LOS by the conjugate are shown in Table 6. 5 μg of OS-TT, OS-HMP, LOS or a mixture of TT and HMP added to 10 ml of female mice (NIH / Swiss) in 0.2 ml of 0.9% NaCl as adjuvant or adjuvant Subcutaneous injection without. The adjuvant used is the same as that used for the dLOS antibody reaction, and the inoculation is performed three times at three week intervals, and blood is collected from the rat at day 14 after the first inoculation, and at the seventh day after the two and three inoculations. It was.

Anti-LOS levels of serum were expressed in ELISA units (EU) using LOS isolated from strain 25238 as coating antigen. For reference, the analysis of the high immune serum of the whole cell of the 25238 strain, 65,000EU / ml for IgG, 800EU / ml for IgM was obtained. Serum antibodies against TT or HMP were measured by ELISA as described for dLOS conjugates. For statistical analysis of these results, antibody levels are expressed as geometric mean ELISA units or n independent observations ± standard deviation or inverse titration of range (n <4). Significance was measured by a two-sided t test and p values less than 0.05 were considered significant.

As shown in the data of Table 6, the non-binding simple mixture of OS, TT and HMP did not form anti-LOS antibodies. The two conjugates formed low levels of anti LOS IgG after two inoculations, but not after one inoculation, and a small increase after three inoculations (p <0.01). OS-TT and OS-HMP showed similar levels of anti-LOS IgG after three inoculations. The LOS alone and the conjugate showed similar levels of anti-LOS IgG after two inoculations, but the LOS alone showed significantly higher anti-LOS IgG than the conjugate after three inoculations. The addition of adjuvant to both conjugates significantly improved their immunogenicity.

Mouse Antibody Response to M. catarrhalis Species 25238 LOS Represented by Conjugates Immunogen ^a Inoculation frequency Geometric mean (± standard deviation range) ^b ELISA units IgG IgM OS-TT1 123 16 (1-30) 4 (1-35) 12 (1-3) 5 (1-46) OS-TT1 + Adjuvant 123 2 (1-3) 4 (1-22) 7 (1-48) 3 (1-9) 3 (1-9) 50 (12-209) OS-TT2 123 118 (1-70) 2 (1-6) 4 (1-14) 7 (2-28) OS-TT2 + Adjuvant 123 1 (1-2) 34 (5-249) 113 (13-957) 2 (1-5) 19 (6-62) 126 (39-409) OS-HMP1 123 12 (1-4) 5 (1-72) 12 (1-5) 4 (1-40) OS-HMP1 + Adjuvant 123 1 (1-2) 290 (5-1,585) 2 (1-9) 3 (1-9) 81 (26-257) OS-HMP2 123 11 (1-2) 3 (1-13) 14 (1-15) 7 (2-25) OS-HMP2 + Adjuvant 123 16 (1-26) 38 (8-176) 7 (2-25) 34 (12-92) 195 (46-828) OS + TT + HMP 123 111 (1-2) 113 (2-5) LOS 123 18 (2-40) 113 (20-630) 3 (1-9) 2 (1-4) 52 (12-230)

a: conjugate of 10 rats of each group by subcutaneous injection three times at three week intervals;

Combined with a Ribi adjuvant; LOS; Or of OS, TT and HMP

The mixture (5 μg each) was inoculated with 5 μg each. Blood samples are inoculated first

After 2 weeks, 1 week after the 2nd and 3rd inoculation.

b: ELISA unit is based on standard serum for strain 25238 and strain

LOS obtained at 25238 was used as coating antigen. Blood before preimmune

Blue contains 1 (1-2) U IgG and 1U IgM.

The mouse antibody response to TT formed by OS-TT conjugates is shown in Table 7. The IgG antibody response was much stronger than the IgM antibody response. Adjuvant enhances the immune response of OS-TT2 but did not have a significant effect on OS-TT1.

Mouse antibody response to TT induced by OS-TT conjugates. Immunogen ^a Inoculation frequency Geometric mean (± standard deviation range) ^b ELISA units IgG IgM OS-TT1 123 2 (1-3) 22 (7-72) 90 (34-237) 111 OS-TT1 + Adjuvant 123 4 (1-9) 126 (69-229) 90 (64-128) 4 (2-6) 5 (3-9) 22 (9-51) OS-TT2 123 95 (33-269) 52 (9-296) 140 (12-1,614) 2 (1-3) 2 (1-4) 5 (2-11) OS-TT2 + Adjuvant 123 113 (66-191) 271 (81-908) 1,756 (454-6,769) 3 (1-5) 19 (9-41) 90 (48-169) OS + TT + HMP 123 14 (8-25) 470 (257-851) 908 (332-2,487) 1 (1-2) 2 (1-6) 14 (5-40)

a: See footnote a in Table 6

b: ELISA unit is based on standard serum for TT and TT is coated antigen

Used as Pre-immune serum has IgG or IgM unit of 1U or less

All.

Mouse antibody responses to HMP induced by OS-HMP conjugates are shown in Table 8. While the level of IgM remained low in the TT antibody response, the level of IgG was significantly increased and the presence of adjuvant increased the IgG antibody response more dramatically than the TT antibody response shown in Table 7. .

Mouse antibody response to HMP formed by OS-HMP conjugates Immunogen ^a Geometric mean (± standard deviation range) ELISA unit ^b Inoculation frequency IgG IgM OS-HMP1 123 365 (40-105) 183 (83-398) 1 (1-2) 14 (5-37) 8 (3-21) OS-HMP1 + Adjuvant 123 9 (2-34) 585 (330-1,442) 1,506 (507-4,464) 2 (1-3) 5 (3-8) 6 (3-12) OS-HMP2 123 9 (4-19) 81 (39-168) 175 (34-902) 3 (2-5) 4 (2-12) 2 (1-6) OS-HMP2 + Adjuvant 123 81 (45-146) 1,131 (642-1,997) 4,228 (2,600-7,454) 6 (3-14) 17 (8-38) 38 (12-120) OS + TT + HMP 123 2 (1-4) 470 (257-851) 1,53 (752-3,281) 1334 (11-104)

a: See footnote a in Table 6

b: ELISA unit is based on standard serum for HMP and HMP is coated antigen

Was used. Preimmune serum contains 1 to 3 units of IgG or IgM

Indicated.

The rabbit antibody response to M. catarrhalis LOS formed by the conjugate is shown in Table 9, and the rabbit antibody response to TT and HMP induced by the conjugate is shown in Table 10.

Rabbit antibody response to M. catarrhalis LOS formed by the conjugate Immunogen ^a Inoculation frequency Geometric mean (± standard deviation range) ELISA unit ^b IgG IgM OS-TT2 012 22 (3-90) 140 (30-810) 7,290 (2,430-21,870) 7 (3-30) 52 (30-90) 68 (30-90) OS-TT2 + Ribi 012 4 (3-10) 729 (270-2,430) 12,627 (7,290-21,870) 10 (3-30) 156 (90-270) 90 OS-HMP2 012 17 (10-30) 467 (90-2,430) 7,290 (2,430-21,870) 7 (3-30) 10 (3-30) 90 (30-270) OS-HMP2 + Ribi 012 9 (3-30) 81012,627 (7,290-21,870) 7 (3-30) 30 (10-90) 118 (90-270) OS + TT + HMP 012 5 (3-10) 5 (3-10) 38 (3-90) 17 (10-30) 5 (3-10) 17 (10-30) LOS 012 6 (3-10) 10 (3-300) 52 (30-90) 6 (3-10) 52 (30-90) 90

a: Subcutaneous and intramuscular injection of 2 to 3 rabbits in each group twice a month

Furnace conjugates; Combined with a Ribi adjuvant; LOS; Or OS, TT and

A mixture of HMP (50 μg each) was inoculated at 50 μg each. Blood samples

Collection was made before inoculation and 14 days after each inoculation.

b: See footnote b in Table 6.

Rabbit antibody response to protein (TT or HMP) formed by the conjugate Immunogen ^a Inoculation frequency Geometric mean (± standard deviation range) ^b ELISA units IgG IgM For the analysis of anti-TT OS-TT2 012 102704,209 (2,430-7,290) 1017 (10-30) 118 (90-270) OS-TT2 + Ribi 012 17 (10-30) 2,43021,870 1090355 (90-810) OS + TT + HMP 012 30468 (270-810) 2,430 17 (10-30) 155 (90-270) 90 For the analysis of anti-HMP OS-HMP2 012 52 (30-90) 118 (90-270) 2,430 17 (10-30) 3052 (30-90) OS-HMP2 + Ribi 012 30468 (270-810) 7,290 17 (10-30) 3030 OS + TT + HMP 012 52 (30-90) 2702,430 17 (10-30) 3052 (30-90)

a: See footnote a in Table 9

b: See footnote b in Tables 7 and 8

In rats, 40% (8 out of 20 rats) of OS immunized with OS protein conjugates and of serum immunized with conjugates (with adjuvant) tripled OS-TT or OS-HMP. After inoculation, bactericidal activity against pure single species M. catarrhalis strain 25238 was found in the range of 1: 8 to 1:64. After two inoculations of OS-TT alone or in combination with an adjuvant, bactericidal activity against strain 25238 of pure single species was shown within the range of 1: 2 to 1: 8.

As described above in detail specific parts of the present invention, for those of ordinary skill in the art, these specific techniques are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (38)

Removing esterified fatty acids to construct detoxified lipoologosaccharides (dLOS) isolated from Moraxella catarrhalis or removing lipid A to construct oligosaccharides (OS, lipoologosaccharides). A conjugate vaccine against Moraxella catarrhalis in which an immunological carrier is covalently bound to lipo-oligosaccharides (LPS) lipo-oligosaccharides (LPS) and electrically non-toxic lipo-oligosaccharides. The method of claim 1, The immunological carrier is a protein, characterized in that Combined vaccine against Moraxella catarrhalis. The method of claim 2, Immune carrier proteins are Moraxella catarrhalis UspA, CD, tetanus toxin / denaturing toxin, isolated from Is isolated from Haemophilus influenzae. Molecular Weight Protein, Diphtheria Toxin / Modified Toxin, Non-Toxic Pseudomonas Aeruginosa Toxin A, cholera toxin / modified toxin, pertussis toxin / modified toxin, clostridium fur Clostridium perfringens exotoxin / modified toxin, hepatitis B surface Antigen, Hepatitis B core antigen, Rotavirus VP7 protein, CRM, CRM ₁₉₇ , From the group consisting of CRM ₃₂₀₁ and respiratory syncope virus F and G white matter Characterized in that selected Combined vaccine against Moraxella catarrhalis. The method of claim 3, wherein Immune carrier proteins are tetanus toxins or high molecular weight proteins By Combined vaccine against Moraxella catarrhalis. A pharmaceutical composition comprising the conjugate vaccine of claim 1 in a pharmaceutically acceptable carrier. The method of claim 5, Characterized in that it further comprises an adjuvant Pharmaceutical compositions. The method of claim 6, Adjuvant is a combination of monophosphate lipid A and trehalose dimycolic acid or alum. Characterized in that the mixture Pharmaceutical compositions. The immunological carrier is a conjugate vaccine, characterized in that it is covalently linked to the dLOS or OS via a linker compound. The method of claim 8, Linked compounds include adipic acid dihydrazide, ε-aminohexane, chlorohexane Consisting of phenol dimethyl acetal, D-glucuronolactone, p-nitrophenyl amine Characterized in that selected from the group Conjugate vaccine. The method of claim 8, Characterized in that the linking compound is adipic acid dihydrazide Conjugate vaccine. Lipooligosaccharide isolated from Moraxella catarrhalis and detoxified by removing ester bound fatty acids. The method of claim 11, Moraxella (Moraxella) with lipooligosaccharides isolated catarrhalis) is purely isolated Moraxella catarrhalis. Characterized Lipooligosaccharides. Lipooligosaccharide isolated from Moraxella catarrhalis and inactivated by removing Lipid A. Effectively immunizing mammals with a non-toxic lipooligosaccharide (dLOS) derived from lipooligosaccharides of Moraxella catarrhalis or a conjugate vaccine comprising an oligosaccharide (OS) and an immunological carrier covalently linked to dLOS A method of preventing otitis media caused by an infection of Moraxella catarrhalis in a mammal, comprising administering in a protectable amount. The method of claim 14, Mammals are human A method of preventing otitis media caused by an infection of Moraxella catarrhalis in a mammal. The method of claim 14, The conjugate vaccine is characterized by administration parenterally. A method of preventing otitis media caused by an infection of Moraxella catarrhalis in a mammal. The method of claim 16, The conjugate vaccine can be injected intramuscularly, subcutaneously, into the mucous membranes of the nose, or Characterized in that administered by their combination A method of preventing otitis media caused by an infection of Moraxella catarrhalis in a mammal. The method of claim 14, An immunoprotective effective amount is about 10-50 μg per dose Characterized by A method of preventing otitis media caused by an infection of Moraxella catarrhalis in a mammal. The method of claim 14, Booster injection with a conjugate of about 10 to 25 μg Characterized in that it further comprises a step How to prevent otitis media caused by infection with Moraxella catarrhalis in mammals The method of claim 14, About 1 to 50 μg of conjugate vaccine was first inoculated and about two months later Next, a second dose of about 10 to 25 μg of the conjugate vaccine is given, followed again by two months. 3 vaccinated with about 10 to 25 μg of conjugate vaccine, After the passage, the procedure of 4th inoculation of the conjugate vaccine Characterized in that the administration step comprises How to prevent otitis media caused by infection with Moraxella catarrhalis in mammals A method for detoxifying a lipooligosaccharide isolated from Moraxella catarrhalis, comprising removal of ester bound fatty acids from lipooligosaccharides isolated from Moraxella catarrhalis. The method of claim 21, Treatment of lipooligosaccharides with hydrazine or weak alkali reagents Characterized in that the bound fatty acids are removed A method of detoxifying lipooligosaccharides (LOS) isolated from Moraxella catarrhalis. (i) removing ester bound fatty acids from lipooligosaccharides isolated from Moraxella catarrhalis; And (ii) a method of preparing a conjugate vaccine against Moraxella catarrhalis for producing non-toxicized LOS (dLOS), comprising covalently binding such non-toxicized lipooligosaccharide to one immunological carrier. The method of claim 23, wherein The removal step involves lipooligosaccharides with hydrazine or weak alkali reagents. Characterized in that it comprises a process of processing Method for preparing a conjugate vaccine against Moraxella catarrhalis. The method of claim 23, wherein The linking step involves linking a non-toxic lipooligosaccharide to a linker compound. Adhering to the linker and binding the linking compound to an immunological carrier Characterized Method for preparing a conjugate vaccine against Moraxella catarrhalis. The method of claim 25, Linker compounds include adipic acid dihydrazide, ε-ami Nucleic acid, chlorohexanol dimethyl acetal, D-glucuronolactone or p-nit Rophenyl amine, characterized in that Method for preparing a conjugate vaccine against Moraxella catarrhalis. The method of claim 25, The linker compound is adipic acid dihydrazide Characterized by Method for preparing a conjugate vaccine against Moraxella catarrhalis. The method of claim 23, wherein Adjuvant is applied to non-toxicized lipooligosaccharide bound to an immune carrier. Characterized in that it further comprises the step of adding Method for preparing a conjugate vaccine against Moraxella catarrhalis. (i) removing Lipid A from lipooligosaccharide (LOS) isolated from Moraxella catarrhalis to construct oligosaccharides; And (ii) covalently binding an oligosaccharide to an immunological carrier. The method of claim 29, The removing step includes treating lipooligosaccharides with an acidic reagent. Characterized by Method for preparing a conjugate vaccine against Moraxella catarrhalis. The method of claim 29, The binding step attaches the oligosaccharides to the linker compound, Attaching the linking compound to an immunological carrier Method for preparing a conjugate vaccine against Moraxella catarrhalis. The method of claim 31, wherein Linker compounds include adipic acid dihydrazide, ε-ami Nucleic acid, chlorohexanol dimethyl acetal, D-glucuronolactone or p-nit Rophenyl amine, characterized in that Method for preparing a conjugate vaccine against Moraxella catarrhalis. The method of claim 32, The linker compound is adipic acid dihydrazide Characterized Method for preparing a conjugate vaccine against Moraxella catarrhalis. The method of claim 29, Adding an adjuvant to the oligosaccharide bound to the immunological carrier further It characterized by including Method for preparing a conjugate vaccine against Moraxella catarrhalis. To prevent otitis media due to infection of Moraxella catarrhalis to a mammal, remove esterified fatty acids to construct a non-toxic lipooligosaccharide (dLOS) isolated from Moraxella catarrhalis, or A conjugate vaccine comprising a lipooligosaccharide (LPS), which is nontoxic by a method of removing lipid A to construct an oligosaccharide (OS), and an immunocarrier covalently bound to an electrically nontoxic, lipooligosaccharide. The method of claim 35, wherein The immunological carrier is a protein, characterized in that Conjugate vaccine. The method of claim 36, UspA, CD, wherein the immunological carrier protein was isolated from Moraxella catarrhalis Tetanus toxin / modified toxin, haemophilus whose type cannot be determined High molecular weight protein isolated from Haemophilus Influenzae, Diphtheria Toxin / Denatured Toxin, Non-Toxic Pseudomonas Aeruginosa Toxin A, Cholera Toxin / Denatured toxin, pertussis toxin / denatured toxin, clostridial perfringens (Clostridium perfringens) exotoxin / modified toxin, hepatitis B surface antigen, B Hepatitis Core Antigen, Rotavirus VP7 Protein, CRM, CRM ₁₉₇ , CRM ₃₂₀₁ and From the group consisting of respiratory syncytial virus F and G proteins Characterized in that selected Conjugate vaccine. The method of claim 37, The immunological carrier is characterized by tetanus toxin or high molecular weight protein. doing Conjugate vaccine.