Patent 6,685,949

A conjugate vaccine for Moraxella (Branhamella) catarrhalis comprising isolated lipooligosaccharide from which esterified fatty acids have been removed, to produce a detoxified lipooligosaccharide (dLOS), or from which lipid A has been removed, to produce a detoxified oligosaccharide (OS), which is linked to an immunogenic carrier. The vaccine is useful for preventing otitis media and respiratory infections caused by M. catarrhalis in mammals, including humans.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is disclosed conjugate vaccine for Moraxella catarrhalis, including a lipooligosaccharide (LOS) isolated from M. catarrhalis and detoxified by treating to remove esterified fatty acids to produce a detoxified LOS (dLOS), or by treating to remove lipid A to produce an oligosaccharide (OS), and an immunogenic carrier covalently linked thereto. In one embodiment, the immunogenic carrier is a protein. In another embodiment, the immunogenic carrier protein is selected from the group consisting of UspA isolated from M. calarrhalis, CD isolated from M. catarrhalis, tetanus toxin/toxoid, a high molecular weight protein (HMP) isolated from nontypeable Haemophilus influenzae, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 protein; CRMs (Cross Reacting Materials), including CPM₁₉₇ (Pappenheimer et al., Immunochem. 9:891-906, 1972) and CRM₃₂₀₁ (Black et al., Science 240:656-659, 1988); and respiratory syncytial virus F and G protein. In one aspect of the vaccine, the immunogenic carrier protein is tetanus toxoid or HMP. Another embodiment is a pharmaceutical composition that includes such a vaccine conjugate in a pharmaceutically acceptable carrier, which may include an adjuvant. Preferably, the adjuvant is an admixture of monophosphoryl lipid A and trehalose dimycolate or alum. In one embodiment, the immunogenic carrier is covalently linked to de-esterified LOS via a linker compound. Preferably, the linker compound is selected from the group consisting of adipic acid dihydrazide, E-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenylethyl amine, and more preferably, the linker compound is adipic acid dihydrazide. In one embodiment, the vaccine further includes an oligosaccharide (OS) isolated from M. catarrhalis by removal of lipid A from LOS, which is covalently linked to an immunogenic carrier.

According to another aspect of the invention, there is disclosed a lipooligosaccharide isolated from Moraxella catarrhalis and detoxified by removal of ester-linked fatty acids therefrom (dLOS), or an oligosaccharide obtained from removal of lipid A from LOS. In one embodiment, the Moraxella catarrhalis from which the lipooligosaccharide is isolated is a purified strain of Moraxella catarrhalis.

According to another aspect of the invention, there is disclosed a method of preventing otitis media caused by infection with Moraxella catarrhalis in a manmmal, including administering to the manmmal an effective immunoprotective amount of the conjugate vaccine that includes a detoxified lipooligosaccharide (dLOS) produced by de-esterification of LOS derived from Moraxella catarrhalis, or an oligosaccharide (OS) produced by removal of lipid A from LOS, and an immunogenic carrier covalently linked to the dLOS or to the OS. In a preferred embodiment, the mammal is a human. In another embodiment, the conjugate vaccine is administered parenterally. In one embodiment, the conjugate vaccine is administered by intramuscular injection, subcutaneous injection, or by deposit on intranasal mucosal membrane or combinations thereof. In another embodiment, the effective immunoprotective amount is between about 10 .mu.g and about 50 .mu.g per dose. The method may also include injecting between about 10 .mu.g and about 25 .mu.g of the conjugate vaccine at about two months and again at about thirteen months after the administering step. In one embodiment, the administering step includes administering a first dose, and then administering a second dose of about 10 .mu.g to about 25 .mu.g of the conjugate vaccine at about two months after the first dose, administering a third dose of about 10 .mu.g to about 25 .mu.g of the conjugate vaccine at about 2 months after the second dose, and administering a fourth dose of about 10 .mu.g to about 25 .mu.g of the conjugate vaccine at about 12 months after the third dose.

According to another aspect of the invention, there is disclosed a method for detoxifying lipooligosaccharide (LOS) isolated from Moraxella catarrhalis, including removing ester-linked fatty acids from the LOS. In one embodiment, the ester-linked fatty acids are removed with hydrazine or a mild alkaline reagent.

The invention also includes a method for detoxifying LOS from Moraxella catarrhalis, including removal of lipid A from the LOS to produce OS. In one embodiment, the lipid A is removed by acid treatment.

According to another aspect of the invention, there is disclosed a method of making a conjugate vaccine against Moraxella catarrhalis including removing ester-linked fatty acids from lipooligosaccharide (LOS) isolated from M. catarrhalis to produce de-esterified LOS (dLOS); and covalently linking the dLOS to an immunogenic carrier.

In one embodiment, the removing step comprises treating the LOS with hydrazine or a mild alkaline reagent. In one embodiment, the linking step includes attaching the dLOS to a linker compound and attaching the linker compound to the immunogenic carrier.

Preferably, the linker compound is adipic acid dihydrazide, .epsilon.-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone or p-nitrophenylethyl amine, and more preferably, the linker compound is adipic acid dihydrazide. In another embodiment, the vaccine composition may include an adjuvant.

The present invention also provides a conjugate vaccine comprising a lipooligosaccharide (LOS) isolated from M. catarrhalis and detoxified by treating to remove esterified fatty acids to produce detoxified LOS (dLOS), or by removing lipid A to produce oligosaccharide (OS), and an immunogenic carrier covalently linked thereto, for use in preventing otitis media caused by infection with Moraxella catarrhalis in a mammal. Preferably, the immunogenic carrier is a protein. In one aspect of this preferred embodiment, immunogenic carrier protein is UspA isolated from M. catarrhalis, CD isolated from M. catarrhalis, tetanus toxin/toxoid, a high molecular weight protein (HMP) isolated from nontypeable Haemophilus influenzae, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 protein; CRMs including CRM₁₉₇ (Pappenheimer et al. supra.) and CRM₃₂₀₁, (Black et al., supra.); or respiratory syncytial virus F and G protein. Preferably, the immunogenic carrier protein is tetanus toxoid or HMP.

DETAILED DESCRIPTION OF THE INVENTION

Lipooligosaccharide (LOS) of Moraxella (Branhamella) catarrhalis is a major surface antigen that elicits bactericidal antibodies against bacteria that cause otitis media and sinusitis in children and respiratory tract infections in adults. For simplicity, the bacteria are referred to hereinafter as Moraxella catarrhalis or M. catarrhalis. The M. catarrhalis LOS was isolated and treated to reduce its toxicity by about 20,000-fold, as assayed using a Limulus amebocyte lysate (LAL) test. The detoxified LOS (dLOS) was coupled to a carrier (e.g., tetanus toxoid or high-molecular-weight proteins purified from nontypeable Haemophilus influenzae) through a linker compound to form dLOS-TT or dLOS-HMP. The molar ratios of dLOS to TT and HMP in the resulting conjugates were about 19:1 and 31:1, respectively. The antigenicity of the two conjugates was similar to that of isolated LOS, as determined by a double-immunodiffusion assay. For both dLOS-carrier conjugates, subcutaneous (s.c.) or intramuscular (i.m.) injection into animals elicited increased mean levels of immunoglobulin G (IgG) to LOS. In mice, a 50- to 100-fold rise in the mean IgG levels was detected after three injections of the conjugates, and in rabbits, a 350- to 700-fold rise of IgG levels was detected after two injections. The immunogenicity of the conjugate was enhanced by inclusion of an adjuvant in the conjugate formulation.

In rabbits, antisera produced after conjugate immunization induced complement-mediated bactericidal activity against the homologous strain and heterologous strains of M. catarrhalis. These results show a detoxified LOS-protein conjugate is useful as a vaccine for immunizing against M. catarrhalis-caused diseases.

A purified type A M. catarrhalis strain (ATCC strain 25238, available from the American Type Culture Collection, Rockville, Md.) was used as an exemplary source for purification of LOS using standard methods (Edebrink, P., et al., 1994, Carbohydr. Res. 257:269-284; Masoud, H., et al., 1994, Can. J. Chem. 72:1466-1477). Other known strains of M. catarrhalis, many of which are available from the ATCC or other repositories, or purified clinical isolates obtained using well known bacteriological methods are also within the scope of the invention for use as a source of LOS. Briefly, the M. catarrhalis strain 25238 was grown on chocolate agar for 8 hr, and then inoculated into 3% tryptic soy broth (TSB) which was incubated with shaking at 37^oC. overnight. The culture was further diluted and transferred to baffled flasks containing TSB, and grown with shaking at 37^oC. for an additional 24 hr. The cells were collected by centrifugation, and the pelleted cells were washed with ethanol, acetone, and petroleum ether using standard methods (as described in Masoud, H., et al., 1994, Can. J. Chem. 72:1466-1477), before being dried to a powder. The LOS was extracted from cells by a standard hot phenol-water method (Westphal, O., et al., 1965, Methods Carbohydr. Chem. 5:83-91) with modifications (Gu, X--X., 1995, Infect. Immun. 63:4115-4120) to yield LOS with a protein and nucleic acid content of less than 1% (Smith, P. K., et al., 1985, Anal. Biochem. 150:76-85; Warburg, O. & W. Christian, 1942, Biochem. Z. 310:384-421). Other known methods of LOS purification may be substituted for the methods described herein.

Although the use of hydrazine for detoxification of LOS from M. catarrhalis is described herein, the use of any reagent or enzyme capable of removing esterified fatty acids from lipid A, such as mild alkaline treatment, i.e., treatment with dilute (0.1 N) NaOH or other dilute aqueous base solutions having a pH of between about 13.2 and 13.6, is within the scope of the present invention. It is important that the detoxification conditions be mild enough to not hydrolyze the oligosaccharide portion of the LOS. Hydrolysis of the oligosaccharide will destroy protective epitopes. The isolated M. catarrhalis LOS was detoxified using anhydrous hydrazine treatment under mild conditions substantially as previously described (Gu, X. X., et al., 1996, Infect. Immun. 64:4047-4053; Gupta, R. K., et al., 1992, Infect. Immun. 60:3201-3208). Briefly, LOS was suspended in anhydrous hydrazine and incubated at a temperature of between 1^oC. and 100^oC., preferably between 25^oC. and 75^oC., and more preferably, about 37^oC. Incubation with mixing was between 10 min to 24 hr, preferably about 2 hr to about 3 hr, and then the mixture was cooled and cold acetone was added until a precipitate formed which was collected by centrifugation. The pellet was washed with acetone, dissolved in water, and then ultracentrifuged. The supernatant obtained after ultracentrifugation was freeze-dried, redissolved and subjected to column chromatography to elute the carbohydrate-containing fractions, which were pooled, freeze-dried, and designated dLOS. By weight, the dLOS was about 38% of LOS.

Alternatively, LOS can be detoxified by mild acid treatment using dilute or weak aqueous acids having a pH of between about 2 and 3, as disclosed by Gu et al. (Infect. Immun. 61:1873-1880, 1993) which results in removal of lipid A to produce an oligosaccharide (OS). This OS is then conjuated to carriers using the same methods as for dLOS. Although the use of acetic acid for removal of lipid A from M. catarrhalis LOS is described herein, the use of any reagent or enzyme capable of removing lipid A is within the scope of the present invention. The OS-protein conjugates are also immunogenic in both mice and rabbits, and elicit antibodies to both LOS and the carrier proteins. In mice, conjugate(with adjuvant)-immunized sera showed bactericidal activity against the homologous M. catarrhalis strain 25238. In rabbits, conjugate-immunized sera showed bactericidal activity against homologous strain 25238.

For preparation of dLOS or OS conjugates, the dLOS may be directly covalently bonded to a carrier protein, for example, by using a cross-linking reagent such as glutaraldehyde. Preferably, the dLOS or OS conjugates are produced by use of a linker compound separating the dLOS or OS and the carrier, using any of a variety of known methods (e.g., see Marburg et al., 1986, J. Am. Chem. Soc. 108:5282, and U.S. Pat. Nos. 4,882,317; 5,153,312; 5,204,098). Presence of a linker promotes efficient coupling of the dLOS or OS to the carrier and optimizes immunogenicity of the conjugate. Linkers having chains whose length and flexibility can be adjusted as desired may separate the carbohydrate and carrier components. Linkers may permit increased translational and rotational characteristics of the conjugate antigens, thus increasing access of the binding sites of antibodies. Between the bifunctional sites, the linker chains may contain a variety of structural features, including heteroatoms and cleavage sites. Although adipic dihydrazide (ADH) is a preferred linker, other suitable linkers include, for example, heterodifunctional linkers such as .epsilon.-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenyl amine. Coupling reagents contemplated for use in the present invention include hydroxysuccinimides and carbodiimides. Many suitable linkers and coupling reagents are known to those of ordinary skill in the art (Dick et al., Conjugate Vaccines, J. M. Cruse & R. E. Lewis, Jr., eds., Karger, N.Y., pp. 48-114, 1989).

In a preferred embodiment, the dLOS or OS was first derivatized with adipic dihydrazide (ADH) which serves as the linker to a protein carrier. Briefly, adipic acid dihydrazide (ADH) was bound to the carboxyl group of Kdo moiety of the dLOS or OS to form AH-dLOS or AH-OS derivatives using known methods (Gu, X. X., & C. M. Tsai, 1993, Infect. Immun. 61:1873-1880). A molar excess of ADH was used to ensure more efficient coupling and to limit dLOS-dLOS coupling. In the reaction mixture, the molar ratio of ADH to dLOS or OS is typically between about 10:1 and about 250:1, preferably between about 50:1 and about 150:1, and more preferably, about 100:1. In a preferred embodiment, one ADH per dLOS or OS is present in the AH-dLOS conjugate. In another preferred embodiment, in the final dLOS-carrier conjugate, the molar ratio of dLOS or OS to carrier is between about 15 and about 100, in a preferred lower range of about 20 to about 35 and a preferred upper range of about 40 to about 75, preferably between about 25 and about 50, and more preferably about 50. This ratio is generally controlled by varying the starting concentrations of AH-dLOS or AH-OS and carrier, and the time of reaction. Generally, within these ranges, there was a positive correlation between the ratio and the antibody response to the conjugate in vivo (i.e., the higher the ratio, the higher the response). The derivatized dLOS or OS was purified from the reaction mixture by column chromatography to obtain eluate fractions containing both carbohydrate and adipic hydrazide (Kemp, A. H. & M. R. A. Morgan, 1986, J. Immunol. Methods 94:65-72). These fractions were pooled, freeze-dried, and designated AH-dLOS or AH-OS.

Although a preferred embodiment of the present invention is M. catarrhalis dLOS or OS linked to a protein, more preferably tetanus toxoid (TT) or high molecular weight proteins (HMP) purified from H. influenzae, a variety of carriers known in the art are also suitable for producing the dLOS- or OS-carrier conjugates of the present invention. HMP refers to a group of surface-exposed high molecular weight proteins that are major antibody targets in human convalescent sera obtained from individuals who have been infected with H. influenzae (further defined structurally and functionally by S. J. Barenkamp, 1992, J. Infect. Dis. 165(Suppl. 1):S181-184). The carrier increases the immunogenicity of the oligosaccharide and antibodies raised against the carrier may be medically beneficial. The carrier may be water soluble or insoluble. Suitable natural or synthetic polymeric immunogenic carriers include, for example, materials containing a primary and/or secondary amino group, an azido group or a carboxyl group.

Any one of a variety of immunogenic carrier proteins may be used to produce the dLOS- or OS-carrier conjugates of the present invention. These proteins include, for example, pili, outer membrane proteins and excreted toxins of pathogenic bacteria, nontoxic or "toxoid" forms of such excreted toxins, nontoxic proteins antigenically similar to bacterial toxins (known as cross-reacting materials or CRMs) and other proteins. Preferred outer membrane proteins are those isolated from gram-negative bacteria. Preferred outer membrane proteins include UspA and CD isolated from M. catarrhalis outer membrane. Toxoids are also preferred. Nonlimiting examples of bacterial toxins and toxoids contemplated for use in the present invention include, for example, tetanus toxin or toxoid, diphtheria toxin or toxoid, detoxified P. aeruginosa toxin A, cholera toxin or toxoid, pertussis toxin or toxoid, and Clostridium sp. exotoxin or toxoid. The use of viral proteins (e.g., hepatitis B surface or core antigens; rotavirus VP 7 protein and respiratory syncytial virus (RSV) F and G proteins) as carriers is also contemplated.

CRMs include CRM₁₉₇, antigenically equivalent to diphtheria toxin (Pappenheimer et al., supra.) and CRM3201, a genetically manipulated variant of pertussis toxin (Black et al., supra.). The use of immunogenic carrier proteins from non-mammalian sources, such as, for example, keyhole limpet hemocyanin, horseshoe crab hemocyanin and plant edestin is also within the scope of the invention.

Many coupling methods are envisioned for producing the M. catarrhalis dLOS-or OS-protein conjugates. For example, as presented herein, dLOS or OS was derivatized with AH and then linked to TT or HMP. Alternatively, another method for producing suitable dLOS- or OS-protein conjugates involves cystamine derivatization of dLOS, by EDC-mediated derivatization, followed by disulfide conjugation to N-succimidyl-3-(2-pyridyldithio) propionate-derivatized protein. Other well-known methods for conjugating oligosaccharides to immunogenic carrier proteins are also within the scope of the invention, as described, for example, in U.S. Pat. No. 5,153,312, U.S. Pat. No. 5,204,098; and European Patents EP 0 497 525; and EP 0 245 045.

AH-dLOS or AH-OS was coupled to tetanus toxoid (TT) or high molecular weight proteins (HMP) from H. influenzae to form conjugates (Gu, X. X., & C. M. Tsai, 1993, Infect. Immun. 61:1873-1880). The molar ratio of AH-dLOS or AH-OS to the protein component in the reaction mixture is typically between about 10:1 and about 250:1, preferably is between about 50:1 and about 150:1, and more preferably, is about 100:1. For example, AH-dLOS, dissolved in water, was mixed with TT or HMP at molar ratios of AH-dLOS to conjugating protein of about 100:1. Then, the pH was adjusted to 5.4+0.2 and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl was added to the stirred reaction mixture for 1 hr to 3 hr. The reaction mixture was adjusted to pH 7.0, centrifuged, and purified by column chromatography. Peaks that contained both protein and carbohydrate were pooled, and designated as dLOS-TT or dLOS-HMP, depending on the protein used in the conjugate. Conjugates were analyzed for their carbohydrate and protein compositions using conventional methods, and dLOS and BSA as standards (Dubois, M., et al., 1956, Anal. Biochem. 28:250-256; Smith, P. K., et al., 1985, Anal. Biochem. 150:76-85).

It will be understood by those skilled in the art that the dLOS or OS coupled to the carrier may have originated with a single M. catarrhalis strain or with a variety of strains, to produce a multivalent mixture. Alternatively, dLOS- or OS-carrier conjugates may be prepared individually using a single source of dLOS or OS for production of a single conjugate, and then different conjugates may be mixed subsequently to produce a vaccine containing more than one type of dLOS- or OS-carrier conjugate. In this way, a vaccine containing one or more of the known antigenic types of M. catarrhalis LOS may be produced.

Purified dLOS was characterized and compared to purified LOS using standard SDS-PAGE and silver staining techniques, substantially as described previously (Tsai, C. M. & C. E. Frasch, 1982, Anal. Biochem. 119:115-119). Aliquots of M. catarrhalis LOS (25, 50, 100 and 200 ng) and dLOS (20 .mu.g) were separated on the gel which also contained, as standards, Salmonella minnesota LPS Ra and Rc. Each of the lanes containing M. catarrhalis LOS showed a single band of M_r about 4,000 (Edebrink, P. et al., 1994, Carbohydr. Res. 257:269-284), while the lane containing 20 .mu.g of dLOS did not show a detectable band at the location of LOS. The lane containing 20 .mu.g of dLOS instead showed a faint blurred band at about the level of the S. Minnesota LPS Ra band. These results showed that the dLOS sample contained less than 0.25% residual M. catarrhalis LOS.

The toxicity of isolated LOS, and dLOS were tested by using the standard Limulus amebocyte lysate (LAL) assay (Hochstein, H. D., et al., 1973, Bull. Parenter. Drug Assoc. 27:139-148). The sensitivity of the LAL assay is 0.2 EU/ml, when a standard available from the U. S. F. D. A. was used. The isolated LOS showed 20,000 EU/.mu.g, whereas the dLOS showed 1 EU/.mu.g, representing a 20,000-fold reduction of toxicity. Preferably, a composition having a reduction of about 500-fold to about 1,000-fold EU/.mu.g or more is used for a vaccine. Such reductions of in vitro toxicity determined using, for example, the LAL assay, correlate with reduced and acceptable levels of in vivo toxicity. In vivo toxicity can be readily determined using standard in vivo pyrogen testing methods (e.g., in rabbits, using doses of 0.1 .mu.g to 1 .mu.g/kg of body weight).

The antigenicity of the dLOS, AH-dLOS and the dLOS-TT and dLOS-HMP conjugates was tested by double immunodiffusion using rabbit hyperimmune serum to M. catarrhalis whole cells (strain 25238). The hyperimmune serum was prepared by standard methods. Briefly, two New Zealand white rabbits (female, 2 to 3 kg each) were injected subcutaneously and intramuscularly twice (both s.c. and i.m. for each injection) at four-week intervals with an emulsion of 10⁹ M. catarrhalis whole cells (strain 25238) and incomplete Freund's adjuvant (at a ratio of 1:1, vol/vol). Blood samples were collected before and two weeks after each injection.

Double immunodiffusion was performed using standard methods in a 0.8% agarose gel in phosphate-buffered saline (PBS, pH 7.4). In this assay, the central well contained the rabbit hyperimmune serum to M. catarrhalis whole cells and the surrounding wells individually contained LOS, dLOS-TT, dLOS-HMP, dLOS, and HMP. The hyperimmune serum reacted with the LOS in the double-immunodiffusion assay, producing a sharp, readily detectable band of precipitation. Similarly, the hyperimmune serum reacted with the dLOS to produce a somewhat broader band of precipitation, showing that the isolated dLOS retained the antigenicity of the isolated LOS. The hyperimmune serum also reacted with the dLOS-TT and dLOS-HMP conjugates, producing an identical band of precipitation when compared to LOS. In contrast, the hyperimmune serum did not react measurably with the isolated HMP.

Antigenicity was also measured using an enzyme linked immunosorbent assay (ELISA), using previously-described methods (Gu, X. X., et al., 1996, Infect. Immun. 64:4047-4053), with some modifications. The ELISA plates were coated with LOS and then blocked with 3% BSA. Then, the ELISA wells were incubated with diluted rabbit serum, before alkaline phosphatase-conjugated goat anti-rabbit IgG and IgM (Sigma) was added. Between all of the steps, the wells were washed copiously with PBS containing a polymeric dispersing agent (0.01% Tween-20). The enzyme substrate was added for 30 min, and then the reactions were quantitated at A₄₀₅. The antigenicity of the dLOS-carrier conjugates was determined similarly, using the conjugates as coating antigens and a diluted rabbit immune serum as a binding antibody. Both dLOS-carrier conjugates showed comparable binding to rabbit hyperimmune serum, and the antigenicity of the dLOS-carrier conjugates was higher than that of LOS under the same conditions.

To determine in vivo antigenicity, the dLOS-carrier conjugates were injected parenterally into mice and rabbits and the levels of anti-LOS antibodies in the animals' sera was measured subsequently using ELISA. In mice, a nonconjugated mixture of dLOS and TT or HMP did not elicit anti-LOS antibodies. In contrast, both dLOS-TT and dLOS-HMP conjugates elicited low levels of anti-LOS IgG after a second injection of the conjugate. Following a third injection of the conjugate, there was about a 50-to 100-fold rise in anti-LOS IgG. Both dLOS-TT and dLOS-HMP elicited similar levels of anti-LOS IgG after three injections. LOS alone and the dLOS-carrier conjugates elicited similar levels of anti-LOS IgG.

Formulation of both dLOS-TT and dLOS-HMP conjugates with an adjuvant significantly enhanced immunogenicity in mice. That is, two doses of the dLOS-carrier conjugates with adjuvant elicited comparable or higher IgG levels than that of three doses of the conjugates alone. After three injections of the dLOS-carrier conjugate with adjuvant, there was about a 9 to 15-fold rise of anti-LOS IgG over the levels obtained following three injections of the same conjugate without adjuvant. The dLOS-TT conjugate elicited lower levels of IgG than did the dLOS-HMP conjugate after three injections of conjugate-adjuvant formulations.

The adjuvant used contained monophosphoryl lipid A and trehalose dimycolate (commercially available as Ribi-700, from Ribi Immunochemical Research, Hamilton, MT). Also contemplated within the scope of the invention are other well known standard adjuvants, such as, for example, aluminum compounds (i.e. alum), chemically-modified lipopolysaccharide, suspensions of killed Bordetella pertussis, N-acetylmuramyl-L-alanyl-D-glutamine and other adjuvants known to one of ordinary skill in the art. Additional adjuvants are described by Warren et al. (Ann. Rev. Biochem. 4:369-388, 1986; New Generation Vaccines, 2nd Edition, Levine, M. M. et al., Eds., Marcel Dekker, Inc., New York, 1997). The use of aluminum compounds is preferred, and adjuvants approved for use in humans are particularly preferred. When the mouse sera were assayed for IgM levels, the conjugates elicited low to medium levels of anti-LOS antibodies after each injection, whereas LOS elicited high levels of anti-LOS IgM after the third injection. The addition of an adjuvant to the dLOS-protein conjugates enhanced the levels of anti-LOS IgM produced after the second injection.

When the mouse sera were similarly assayed for antibodies directed against the protein components (TT or HMP) of the dLOS-protein conjugates, anti-protein antibodies were found. For anti-TT antibodies, dLOS-TT elicited low level of IgG after the first injection, and that level rose significantly after the second and third injections. Injection of the dLOS-TT conjugate with adjuvant enhanced the level of IgG produced in dLOS-TT injected group compared to the mice that received with the same conjugate without adjuvant. The unconjugated mixture of TT and dLOS elicited higher levels of anti-TT IgG than that elicited by dLOS-TT. All immunogens elicited low levels of anti-TT IgM, which was increased by the inclusion of adjuvant in the injections.

When the mouse sera were similarly assayed for anti-HMP antibodies, dLOS-HMP elicited a low level of IgG after the first injection, and the anti-HMP IgG level rose significantly after the second and third injections. Inclusion of an adjuvant enhanced the levels of IgG in mice that received the dLOS-HMP conjugate. The unconjugated mixture of HMP and dLOS elicited higher levels of anti-HMP IgG than that seen in the mice that received the dLOS-HMP, with or without adjuvant (see Table 2 below). All of the immunogens elicited low levels of anti-HMP IgM.

Immunogenicity of the dLOS-protein conjugates was also determined for rabbits injected s.c. and i.m. at time 0 and one month later (injections were both s.c. and i.m. for each injections). Blood samples were collected at time 0 (i.e., at the first injection time), two weeks after the first injection, and two weeks after the second injection. Using the ELISA methods as used to measure antigenicity of the mouse sera, the levels of IgM and IgG were determined for rabbit sera obtained after injection with the following inmmunogens (all at 50 .mu.g per immunogen per injection): LOS, dLOS-TF, dLOS-TT with adjuvant, dLOS-HMP, dLOS-HMP with adjuvant, an unconjugated admixture of dLOS, TT, and HMP, or whole M. catarrhalis cells. The mixture of dLOS, TT, and HMP or LOS alone elicited low levels of anti-LOS IgG or IgM antibodies after two injections. The dLOS-TT conjugate elicited a significant rise of anti-LOS IgG after the first and second injections compared to pre-injection serum levels. Injection of the dLOS-HMP conjugate elicited lower levels of IgG than did the dLOS-TT conjugate. Inclusion of an adjuvant enhanced the levels of anti-LOS IgG for both conjugates after each injection, and there was no significant difference between the two conjugates after two injections with adjuvant. For IgM, both conjugates elicited low to medium levels of anti-LOS antibodies, and inclusion of an adjuvant elicited generally increased levels of anti-LOS IgM antibodies detected after each injection, compared to the same conjugate injected without adjuvant.

When the rabbit sera were similarly assayed for antibodies directed against the protein components of the dLOS-protein conjugates, anti-protein antibodies were found. For anti-TT antibodies, dLOS-TT elicited low levels of IgG after the first injection, and the level rose significantly after the second injection. Significantly more anti-TT IgG detected after injection of dLOS-TT with adjuvant, compared to injection without adjuvant. Injection of a nonconjugated mixture of TT, dLOS and HMP elicited higher levels of anti-TT IgG than that elicited by dLOS-TT, but lower than elicited by dLOS-TT with adjuvant after the second injection. All immunogens elicited low to medium levels of anti-TT IgM.

For anti-HMP antibodies found in rabbit sera, dLOS-liMP elicited low level of IgG after the first injection, that level rose significantly after the second injection. Inclusion of an adjuvant with dLOS-HMP enhanced the levels of ant-HMP IgG antibodies. The admixture of HMP, dLOS and TT elicited higher level of anti-HMP IgG than injection of dLOS-HMP without adjuvant, but somewhat less than the dLOS-carrier conjugate with adjuvant. All immunogens elicited low to medium levels of anti-HMP IgM.

The antisera produced in mice and rabbits was assayed for bactericidal activity in vitro against homologous and heterologous strains of M. catarrhalis, using standard methods (Gu X--X., et al., 1996, Infect. Immun. 64:4047-4053). In the rabbit model, sera produced after immunization with LOS or unconjugated dLOS showed no bactericidal activity against the homologous M. catarrhalis strain. In contrast, sera produced in response to immunization with dLOS-TT showed bactericidal activity at mean titers of 1:16 (without adjuvant) and 1:40 (with adjuvant), and sera produced following immunization with dLOS-HMP showed bactericidal activity at mean titers of 1:10 (without adjuvant) and 1:40 (with adjuvant). The anti-LOS IgG levels, as determnined by ELISA, correlated with the detected bactericidal titers.

The bactericidal activities of the antisera against the homologous M. catarrhalis strain and heterologous strains from different geographic areas (e.g., Japan) showed that rabbit sera produced in response to the dLOS-protein conjugates had more bactericidal activity than did similarly produced mouse sera. All of the conjugate-induced rabbit sera showed bactericidal activity against the homologous M. catarrhalis strain and representative sera showed bactericidal activity against most nonhomologous strains (9 of 10 ATCC strains and clinical isolates). In contrast, less than half of the dLOS-carrier conjugate-induced mouse antisera showed bactericidal activities against the homologous strain. Generally, the bactericidal titers and the levels of anti-LOS IgG antibody correlated.

The bactericidal activities of the rabbit antisera elicited by dLOS-TT formulated with an adjuvant were analyzed using twenty additional M. catarrhalis strains (ten wild type ATCC strains and ten clinical isolates). Ten of twenty strains were either complement sensitive or serum sensitive. Using the remaining ten strains, the rabbit antisera demonstrated bactericidal activities to four ATCC and five clinical isolates at the mean titer of 1:15 (range 1:2 to 1:32). One strain was negative in the bactericidal assay.

In the mouse model, 20% (4 of 20 mice) of sera from mice immunized with the dLOS-protein conjugates, and 45% (9 of 20 mice) of sera produced after immunization with conjugates and adjuvant, showed low titers of bactericidal activity against the homologous M. catarrhalis strain (ATCC 25238) after three injections of dLOS-carrier conjugate.

The results presented in the examples that follow show that, after detoxification, M. catarrhalis dLOS retained antigenic determinants but was not immunogenic in vivo. When dLOS was conjugated to protein carriers, the dLOS component become immunogenic. That is, the M. catarrhalis dLOS-carrier conjugates induced significant IgG antibody responses to LOS in mammals. In mammals, the dLOS-carrier conjugates elicited at least similar levels of anti-LOS IgG antibodies as did the LOS. The immunogenicity of the dLOS-protein conjugates was better in rabbits than in mice (i.e., after two injections of the conjugates into rabbits, the fold increase of anti-LOS antibodies was generally higher than the fold increase of anti-LOS antibodies seen in mice after two or three injections of the same conjugates). In both species, the levels of anti-LOS antibodies were enhanced when the conjugate was injected with adjuvant compared to injection of the same conjugate without adjuvant. Both the dLOS-TT and the dLOS-HMP conjugates elicited similar levels of anti-LOS IgG antibodies, which were increased when the conjugates were formulated with an adjuvant.

The M. catarrhalis dLOS-carrier conjugates of the present invention are useful as a vaccine to induce immunity against M. catarrhalis infections in mammals, particularly for preventing otitis media and respiratory diseases in humans. The methods of producing such dLOS-carrier conjugates as disclosed herein are useful for the manufacturing of such vaccines. The methods disclosed herein are also useful for identifying other dLOS-carnier conjugates (i.e., conjugates of dLOS with other carrier moieties) that are useful for inducing protective immune responses to M. catarrhalis in mammals, particularly in humans, including children. It will be understood that a vaccine against M. catarrhalis may include dLOS-carrier conjugate, along with other components, such as immunogenically inert pharmaceutically acceptable agents or clinically acceptable adjuvant. It will also be appreciated by those skilled in the art that M. catarrhalis dLOS-carrier conjugate may also be combined with other immunogenically active components directed against other infectious agents (e.g., to produce a combination vaccine against M. catarrhalis and one or more other bacteria or virus that causes childhood disease); for example, a trivalent vaccine against M. calarrhalis, nontypeable Haemphilus influenzae and Streptococcus pneumoniae to prevent bacterial otitis media.

For vaccination, the dLOS-carrier conjugates are parenterally administered. Although various routes of vaccine administration including, for example, intramuscular (i.m.), subcutaneous (s.c.), intraperitoneal (i.p.), transmucosal (e.g., intranasally) and intraarterial are contemplated, transmucosal, s.c. and i.m. administration are preferred. For parenteral administration, the dLOS-carrier conjugates may be in the form of a sterile preparation, such as, for example, a sterile injectable aqueous or oleaginous suspension, with or without an adjuvant. Such suspensions are formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The sterile preparation may also be a sterile injectable solution or suspension in a parenterally acceptable diluent or solvent, such as, for example, a solution in 1,3-butanediol. Other suitable diluents include, for example, water, Ringer's solution and isotonic sodium chloride solution. Also, sterile fixed oils may be employed conventionally as a solvent or suspending medium. For example, any bland fixed oil may be employed, including synthetic mono- or diglycerides. Also, fatty acids such as oleic acid may likewise be used in the preparation of injectable preparations. For intranasal administration, the formulation may be aerosolized using an inert carrier (e.g., air or hydrocarbon) using any of a variety of conventional methods.

The dLOS-carrier conjugates in a vaccine of the present invention may be in soluble or microparticular form, or may be incorporated into microspheres or microvesicles, including liposomes. In one embodiment, the dosage of the conjugate administered will range from about 10 .mu.g to about 100 .mu.g, preferablv, between about 20 .mu.g and about 50 .mu.g. In another preferred embodiment, the amount administered is about 25 .mu.g to about 40 .mu.g. The exact dosage can be determined by routine dose/response protocols known to one of ordinary skill in the art, generally with doses administered on the basis of body weight, particularly for children.

The vaccine of the invention may be administered to manmmals of any age and are adapted to induce active immunization in young mammals, particularly humans, against otitis media and respiratory infections caused by M. catarrhalis. As a childhood vaccine, the dLOS-carrier conjugate is administered at about two to twelve months of age, preferably between about two to six months of age. Booster injections will likely be given. Typically, two booster injections of between about 10 .mu.g and about 25 .mu.g are administered, for example, at about two months and about thirteen months after the initial injection. Alternatively, booster injections are given at two, four and sixteen months after the initial injection. Other booster injection protocols are also contemplated.

Vaccine compositions may comprise a cocktail of conjugates from different M. catarrhalis strains that protects against all or most medically relevant strains. There are three known types of M. catarrhalis based on dLOS: Types A, B and C which represent 61%, 29% and 5% of clinical isolates, respectively. As shown in Example 6, antisera raised against one strain cross-reacts with some, but not all, other strains. Thus, a cocktail of different conjugates will likely be used. Mixtures of conjugates containing dLOS or OS from Types A and B will cover 90% of all medically relevant strains, while mixtures of conjugates containing dLOS or OS from Types A, B and C will cover 95% of all medically relevant strains.

A passive protection study was performed in mice immunized with either rabbit antisera against dLOS-TT, and then challenged with M. catarrhalis strain 25238 by aerosol chamber. Significant reductions in bacterial CFU per lung were observed in the vaccine group. This mouse pulmonary clearance model mimics the natural transmission of the bacteria in humans. The advantages of this model are that it is simple, repeatable and well controlled by the aerosol machine, large numbers of mice can be studied under the same challenge conditions and there is no surgically invasive procedure required for the inoculation of bacteria.

Although not wishing to be bound to a particular mode of action or mechanism, bactericidal antibodies elicited in response to the dLOS-carrier conjugates, particularly IgG, may transude to mucosal surfaces of nasopharynges. There, the antibodies can inactivate a M. catarrhalis innoculum on the mucosal surface, thus preventing or relieving symptoms of M. catarrhalis-caused otitis media and respiratory diseases. Secretory IgA may also play a role in respiratory mucosal immunity, particularly if the conjugate vaccine is administered to the nasal mucosa.

Claim 1 of 12 Claims

What is claimed is:

1. An immunogenic composition comprising a lipooligosaccharide (LOS) isolated from Moraxella catarrhalis and detoxified by treating to remove esterified fatty acids to produce detoxified LOS (dLOS) and an immunogenic carrier covalently linked thereto.

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