CONJUGATE IMMUNOGENIC COMPOSITIONS
Cross Reference to Related Applications
[0001 1 This application claims the priority benefit of U.S. Provisional Patent Application Nos. 61/219, 143 and 61 /219, 151 , filed June 22, 2009, the entire disclosures of which are each hereby incorporated by reference herein in their entirety.
Field of the Invention
[0002] The invention relates generally to Staphylococcus aureus serotype 5 and 8 capsular polysaccharide conjugate immunogenic compositions and methods for their preparation and use.
Background of the Invention
[0003] Humans are a natural reservoir for Gram-positive Staphylococcus aureus. For example, S. aureus can colonize the skin, nares and throat, either permanently or transiently, without causing disease. S. aureus infections range from mild skin infections to endocarditis, osteomyelitis, bacteremia and sepsis. S. aureus also causes a majority of nosocomial infections, and its prevalence in community-onset infections is increasing. Moreover, in 2005, methicillin-resistant S. aureus

(MRSA) infections were estimated at 3 1 .8 per 100,000 individuals, including 16,650 deaths in the United States in 2005 ( levens el al. (2007) J. Am. Med.
Assoc. 298: 1763- 1 771 ). Disease subsequently occurs when individuals become immunocompromised due to breaches in immune barriers, such as during surgery, placement of indwelling catheters or other devices, trauma or wounds. .
|0004] S. aureus produces a large number of extra- and intra-cellular antigens, including numerous toxins and enzymes. Of particular interest herein are capsular polysaccharide serotypes of S. aureus (see, arakawa & Vann, "Capsular polysaccharides of Staphylococcus aureus " In: Weinstein & Fields, eds. Seminars in Infectious Disease. IV. Bacterial Vaccines. (New York, NY; Thieme Stratton; 1982. pp. 285-293), especially serotype 5 and 8 capsular polysaccharides.
Epidemiological studies on a large number of strains of S. aureus isolated from individuals showed that 70% to 80% were either serotype 5 or 8 capsular polysaccharide (Arbeit et al. ( 1984) Diagn. Microbiol. Infect. Dis. 2:85-91 ).
Unfortunately, the capsular polysaccharides are poor immunogens by themselves. |0005] Staphylococcal infections and diseases dramatically increased in the last twenty years, as has use of intravascular devices and invasive procedures. The rise in disease incidence is more troubling because of a parallel rise of antibiotic resistance; therefore, there is an urgent need for immunogenic compositions to prevent Staphylococcal infect ions and diseases.

SUMMARY OF THE INVENTION

[0006] The present invention is directed towards immunogenic conjugates comprising a S. aureus serotype 5 or 8 capsular polysaccharide conjugated to a carrier protein, and methods for making such conjugates. S. aureus serotype 5 or 8 capsular polysaccharides may be obtained directly from the bacteria using isolation procedures known to those skilled in the art, may be produced using synthetic protocols, or may be recombinantly produced using genetic engineering procedures also known to those skilled in the art. In addition, the present invention provides methods for inducing an immune response against a Staphylococcus bacterium, methods for preventing a disease caused by a Staphylococcus bacterium, and methods for reducing the severity of at least one symptom of a disease caused by infection with a Staphylococcus bacterium.

|0007| In one embodiment, the invention comprises an immunogenic polysaccharide-protein conjugate comprising an isolated Staphylococcus aureus serotype 5 or 8 capsular polysaccharide conjugated to a carrier protein, wherein the polysaccharide has a molecular weight of between 20 kDa and 1000 kDa. In some embodiments, the immunogenic conjugate has a molecular weight of between 200 kDa and 5000 kDa. In one embodiment the polysaccharide portion of the immunogenic conjugate has a molecular weight range of between 70 kDa and 300 kDa. In one embodiment the immunogenic conjugate has a molecular weight range of between 500 kDa and 2500 kDa.

|0008] In one embodiment, the serotype 5 or 8 capsular polysaccharide has a degree of O-acetylation between 10- 100%. In one embodiment, the degree of O-acetylation is between 50- 100%. In one embodiment, the degree of O-acetylation is between 75- 100%. In one embodiment, the immunogenic conjugate generates an antibody that is functional as measured by killing bacteria in either an animal efficacy model or via an opsonophagocytic killing assay.

[0009) In one embodiment, the immunogenic conjugate carrier protein comprises CRM 197. In one embodiment, the CRM 197 is covalently linked to the

polysaccharide through a carbamate linkage, an amide linkage, or both. In one embodiment, the molar ratio of conjugated lysines to CRM 197 can be about 10: 1 to about 25: 1. In one embodiment, the conjugate comprises one covalent linkage between CRM 1 7 and polysaccharide for at least every 5 to 10 saccharide repeat units of the polysaccharide. In one embodiment, the linkage between carrier protein and polysaccharide occurs for once in every 5 repeat units of the polysaccharide.

|0010] In one embodiment, the immunogenic conjugate comprising CRM 197 comprises 5 to 22 lysines or 8 to 1 5 lysines covalently linked to the polysaccharide. In one embodiment, the immunogenic conjugate comprising CR 197 comprises 5 to 23 lysines or 8 to 12 lysines covalently linked to the polysaccharide.

(001 1 ] In one embodiment, the immunogenic conjugate comprises a type 5 or 8 polysaccharide that is 10- 100% O-Acetylated. In one embodiment, the immunogenic conjugate comprises a type 5 or 8 polysaccharide that is 50- 100% O-Acetylated. In one embodiment, the immunogenic conjugate comprises a type 5 or 8 polysaccharide that is 75- 100% O- Acetylated. In some embodiments,

the immunogenic composition can be used to generate antibodies that are functional in an animal efficacy model or an opsonophagocytic killing assay.

[0012] In one embodiment, the immunogenic conjugate comprises less than about 30% free type 5 or 8 polysaccharide compared to the total amount of type 5 or 8 polysaccharide. "

[0013] In one embodiment, the immunogenic conjugate comprises less than about 20% free type 5 or 8 polysaccharide compared to the total amount of type 5 or 8 polysaccharide.

[0014] In one embodiment, the invention comprises an immunogenic composition comprising an immunogenic conjugate as described herein and at least one of an adjuvant, diluent, or carrier.

J00151 The adjuvant can be an aluminum-based adjuvant, such as one or more of aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one embodiment the adjuvant comprises aluminum phosphate.

[0016] In one embodiment, the immunogenic composition comprises less than about 30% free type 5 or 8 polysaccharide compared to the total amount of type 5 or 8 polysaccharide.

|0017] In one embodiment, the immunogenic composition comprises less than about 20% free type 5 or 8 polysaccharide compared to the total amount of type 5 or 8 polysaccharide.

[0018] In one embodiment, the invention comprises a method of inducing an immune response to a Staphylococcus aureus serotype 5 or 8 capsular polysaccharide conjugate in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition as described herein.

[0019] In one embodiment, the invention comprises a method of producing an immunogenic polysaccharide-protein conjugate comprising an isolated

Staphylococcus aureus serotype 5 or 8 capsular polysaccharide conjugated to a can-ier protein, the method comprising the steps of: reacting an isolated S. aureus serotype 5 or 8 capsular polysaccharide with 1 , 1 -carbonyl-di-( 1 ,2,4-triazole)

(CDT) in an organic solvent to produce an activated serotype 5 or 8

polysaccharide; and reacting the activated serotype 5 or 8 polysaccharide with a carrier protein in an organic solvent to produce a serotype 5 or 8

polysaccharide arrier protein conjugate. .

100201 In one embodiment, the method of activating Staphylococcus aureus serotype 5 or 8 capsular polysaccharide further comprises lyophilizing the isolated serotype 5 or 8 polysaccharide and re-suspending the lyophilized polysaccharide in an organic solvent. In one embodiment, the resuspended polysaccharide is activated and then directly reacted with the carrier protein. In one embodiment, the activated isolated serotype 5 or 8 polysaccharide is isolated prior to reacting with the carrier protein. In one embodiment, the isolated activated isolated

serotype 5 or 8 polysaccharide is lyophilized to produce a lyophilized activated isolated serotype 5 or 8 polysaccharide prior to reacting the polysaccharide with carrier protein. In one embodiment, the method of producing an isolated polysaccharide-carrier protein conjugate comprises a step of lyophilizing the carrier protein to produce a lyophilized carrier protein prior to reacting the carrier protein with the polysaccharide. In one embodiment the method of producing an isolated polysaccharide-carrier protein conjugate comprises the step of rc-suspending lyophilized activated isolated serotype 5 or 8 polysaccharide and lyophilized carrier protein in an organic solvent as part of the reaction of the activated isolated serotype 5 or 8 polysaccharide with a carrier protein.

[00211 In one embodiment the method of producing an isolated S. aureus type 5 or 8 capsular polysaccharide-carrier protein conjugate comprises the step of dilut ing the reaction mixture of activated polysaccharide and carrier protein and maintaining a pH of about 8.8 to about 9.2 for at least 4 hours at about 20°C to about 26°C.

[0022] In one embodiment, the reaction mixture of activated 5. aureus

type 5 or 8 capsular polysaccharide and carrier protein is maintained at a pH of about 9.0 for at least 4 hours at about 23°C.

[0023] In one embodiment the method of producing an isolated S. aureus type 5 or 8 capsular polysaccharide-carrier protein comprises the step of isolating the isolated serotype 5 or 8 polysaccharide-protein conjugate after it is produced.

|0024] In one embodiment, the organic solvent used in the method of producing an isolated S. aureus type 5 or 8 capsular polysaccharide-carrier protein conjugate is a polar aprotic solvent. In one embodiment, the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), In one embodiment, the method of producing an isolated polysaccharide-carrier protein conjugate the organic solvent is DMSO.

[00251 1° one embodiment, the method of producing isolated S. aureus type 5 capsular polysaccharide-carrier protein conjugate comprises the step of adjusting the water concentration of the reaction mixture comprising type 5 capsular polysaccharide and CDT in an organic solvent to between about 0.1 and 0.3%. In one embodiment, the water concentration of the reaction mixture comprising type 5 capsular polysaccharide and CDT in an organic solvent is adjusted to about 0.2%.

j 00261 In one embodiment, the step of activating isolated S. aureus type 5 capsular polysaccharide comprises reacting the polysaccharide with about an amount of CDT that is 20 molar excess to the amount of polysaccharide present in the reaction mixture comprising type 5 capsular polysaccharide and CDT in an organic solvent.

|0027| In one embodiment, the method of producing isolated S. aureus type 8 capsular polysaccharidexarrier protein conjugate comprises the step of determining the water concentration of the reaction mixture comprising type 8 capsular polysaccharide. In one embodiment, the amount of CDT added to the reaction mixture to activate the polysaccharide is provided in about an amount of CDT that is cquimolar to the amount of water present in the reaction mixture comprising type 8 capsular polysaccharide and CDT in an organic solvent.

[0028] In one embodiment, the amount of CDT added to the reaction mixture to activate the polysaccharide is provided in about an amount of CDT that is at a molar ratio of about 0.5 : 1 compared to the amount of water present in the reaction mixture comprising type 8 capsular polysaccharide and CDT in an organic solvent. In one embodiment, the amount of CDT added to the reaction mixture to activate the polysaccharide is provided in about an amount of CDT that is at a molar ratio of 0.75: 1 compared to the amount of water present in the reaction mixture comprising type 8 capsular polysaccharide and CDT in an organic solvent.

[0029] In one embodiment, the method which comprises the step of isolating the activated polysaccharide comprises the step of diafiltration.

[0030| In one embodiment, the method which comprises lyophilization of the carrier protein, prior to lyophilization the carrier protein is diaiiltered against NaCl and the w/w ratio of NaCl/protein carrier protein is adjusted to about 0.5 to about 1.5. In one embodiment, the ratio of NaCl to carrier protein is about 1.

[0031 ] In one embodiment, the carrier protein used in the method of producing an isolated S. aureus type 5 or 8 capsular polysaccharide-carrier protein conjugate comprises CRM

[0032] In one embodiment, the CRM used in the method of producing an isolated S. aureus type 5 or 8 capsular polysaccharide-carrier protein conjugate is reacted with the activated serotype 5 or 8 polysaccharide at a ratio by weight of about 1 : 1 .

[0033] In one embodiment , the method of producing an isolated S. aureus type 5 or 8 capsular polysaccharide-carrier protein conjugate comprises the step of mixing the type 5 or 8 capsular polysaccharide with imidazole or triazole prior to mixing with CDT in an organic solvent.

[0034] In one embodiment, the method of producing an isolated S. aureus type 5 or 8 capsular polysaccharide arrier protein conjugate comprises the step of hydrolyzing the serotype 5 or 8 polysaccharide-carrier protein conjugate to remove unrcacted activation groups.

[0035] In one embodiment, the invention provides a method of producing an immunogenic conjugate comprising an isolated Staphylococcus aureus serotype 5 or 8 capsular polysaccharide conjugated to a carrier protein, the method comprising the steps of: reacting a S. aureus serotype 5 or 8 capsular

polysaccharide with 3-(2-pyridyldithio)-propionyl hydrazide (PDPH) and a carbodiimide in an organic solvent to produce a PDPH-linked polysaccharide; reacting the PDPH-linked polysaccharide with a reducing agent to produce an activated polysaccharide; isolating the activated serotype 5 or 8 polysaccharide to produce an isolated activated serotype 5 or 8 polysaccharide; providing an activated carrier protein; reacting the isolated activated serotype 5 or 8 polysaccharide with the activated carrier protein to produce a serotype 5 or 8

polysaccharide-carrier protein conjugate; whereby an immunogenic conjugate comprising an isolated S. aureus serotype 5 or 8 capsular polysaccharide conjugated to a carrier protein is produced. In one embodiment, the activated carrier protein is isolated prior to reacting the activated carrier protein with the activated polysaccharide.

[0036] In one embodiment, the step of isolating the activated carrier protein further comprises lyophilizing the isolated activated serotype 5 or 8 polysaccharide to produce a lyophilized activated serotype 5 or 8 polysaccharide.

|0037] In one embodiment, the bromoacetic acid is a N-hydroxysuccinimide ester of bromoacetic acid (BAANS).

[00381 'n one embodiment, the method of producing serotype 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH comprises the use of an organic solvent that is a polar aprotic solvent. In one embodiment, the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide,

/V-methyl-2-pyrrolidone, and hexamethylphosphoramide (Ή ΡΑ), In one embodiment, the organic solvent is dimethyl sulfoxide ( DMSO).

[0039] In one embodiment, the carbodiimide used in the method of producing serotype 5 or 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH is l -Ethyl-3-(3-dimethylaminopropyl)-carbodiimide ( EDAC).

[0040| In one embodiment, the method of producing serotype 5 or 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH and EDAC comprises the step of reacting the serotype 5 or 8 capsular polysaccharide with PDPH and EDAC in an organic at a polysaccharide:PDPH:EDAC ratio by weight of about 1 :5:3.

|00411 In one embodiment, the reducing agent used in the method of producing serotype 5 or 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH and EDAC is dithiothreitol (DTT).

[0042) In one embodiment, activation of the carrier protein in the method of producing serotype 5 or 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH and EDAC comprises reacting the carrier protein with a bromoacetic acid.

[0043] In one embodiment, the step of isolating the activated serotype 5 or 8 polysaccharide in the method of producing serotype 5 or 8 capsular

polysaccharide-carrier protein conjugate which utilizes PDPH and EDAC comprises diafiltration.

[0044] In one embodiment, the method of producing serotype 5 or 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH and EDAC comprises the step of hydrolyzing the serotype 5 or 8 polysaccharide-carrier protein conjugate to remove unreacted activation groups. In one embodiment, the step of hydrolyzing the serotype 5 or 8 polysaccharide-carrier protein conjugate comprises the addition of cysteamine hydrochloride.

[0045] In one embodiment, the method of producing serotype 5 or 8 capsular polysaccharidc-carricr protein conjugate which utilizes PDPH and EDAC further comprises isolating the immunogenic conjugate comprising an isolated S. aureus serotype 5 or 8 capsular polysaccharide conjugated to a carrier protein.

[0046] In one embodiment, the isolation of the serotype 5 or 8

polysaccharide-carrier protein conjugate comprises diafiltration.

[0047] In one embodiment, the carrier protein used in the method of producing serotype 5 or 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH and EDAC comprises CRM| 97-[0048] In one embodiment, the CRM 197 in the method of producing serotype 5 or 8 capsular polysaccharide-CRM i 97 conjugate which utilizes PDPH and EDAC is added in a ratio by weight of about 1 : 1 CRM ^ apsular polysaccharide molecu le.

|0049| In one embodiment, activated type 5 or 8 capsular polysaccharide used in the method of producing serotype 5 or 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH and EDAC has a size between about 50 kd and about 500 kd.

|0050] In one embodiment, immunogenic conjugate produced in the method of producing serotype 5 or 8 capsular polysaccharide-carrier protein conjugate which utilizes PDPH and EDAC has a. size between about between 400 kd and about

5000 kd.

[0051 ] In one embodiment, the invention provides an immunogenic composition comprising a type 5 or 8 capsular polysaccharide-carrier protein conjugate produced by any of the methods described herein.

[0052] In one embodiment, the invention provides an immunogenic composition comprising a type 5 or 8 capsular polysaccharide-carrier protein conjugate produced by any of the methods described herein and at least one of an adjuvant, diluent or carrier. In one embodiment, the immunogenic compositions comprise an aluminum based adjuvant that can be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one embodiment, the immunogenic compositions described herein comprise the adjuvant aluminum phosphate.

|0053| The immunogenic compositions described herein can comprise less than 30% and less than 20% free type 5 or 8 polysaccharide compared to the total amount of type 5 or 8 polysaccharide. The immunogenic compositions described herein can be stored in water or a low ionic strength neutral pH buffer.

[0054] In one embodiment, the invention provides a method of reducing or preventing a Staphylococcal infection, disease or condition associated with a

Staphylococcus bacteria in a subject, the method comprising the step of administering a therapeutically or prophylactically amount of an immunogenic composition as described herein to the subject. In one embodiment the infection, disease or condition is selected from the group consisting of invasive

Staphylococcus aureus, sepsis and carriage.

[0055] In one embodiment, the invention provides a method of reducing or preventing a Staphylococcal infection in a subject undergoing a surgical procedure, the method comprising the step of administering a prophylacticaUy effective amount of an immunogenic composition as described herein to the subject prior to the surgical procedure.

[0056] In one embodiment, the method of the invention comprises the substitution of CDI for CDT.

|0057| In one embodiment, the invention provides a Staphylococcus aureus

Type 5 or 8 capsular polysaccharide having a molecular weight of between 50 kDa and 800 kDa covalently bound to a carrier protein; wherein the combined

molecular weight of the polysaccharide covalently bound to the carrier protein is between about 400 kDa and 5000 kDa.

|0058| In one embodiment, the polysaccharide covalently bound to carrier protein comprises a polysaccharide portion that has a molecular weight range of between 70 kDa and 300 kDa. In one embodiment, the polysaccharide covalently bound to carrier protein has a molecular weight range of between 500 kDa and 2500 kDa.

|0059| In one embodiment, the carrier protein portion of the polysaccharide covalently bound to carrier protein comprises CRM 197. In one embodiment the CRM 197 is covalently linked to the polysaccharide through a carbamate linkage, an amide linkage, or both. In some embodiments, the molar ratio of conjugated lysines to CRM 197 is about 10: 1 to about 25: 1. In some embodiments,

the polysaccharide covalently bound to carrier protein comprises at least one covalent linkage between CRM 197 at least at every 5 to 10 saccharide repeat units of the polysaccharide. In some embodiments, the polysaccharide covalently bound to carrier protein comprises at least one linkage between CRM 19 and

polysaccharide occurs at every 5 saccharide repeat units of the polysaccharide. In some embodiments, the CRM 197 portion of the polysaccharide covalently bound to the CRM 197 comprises 5 to 22 lysines covalently linked to the polysaccharide. In some embodiments, the CRM197 portion of the polysaccharide covalently bound to the CRM 1 7 comprises 5 to 23 lysines covalently linked to the polysaccharide. In some embodiments, the CRM 19 portion of the polysaccharide covalently bound to carrier protein of comprises 8 to 1 5 lysines covalently linked to the polysaccharide. In some embodiments, the CRM 197 portion of the polysaccharide covalently bound to carrier protein of comprises 8 to 12 lysines covalently linked to the

polysaccharide.

[0060] In one embodiment the invention provides an immunogenic composition comprising a 5. aureus type 5 or 8 polysaccharide covalently bound to carrier protein as described herein and at least one of an adjuvant, diluent, or carrier. |00611 In one embodiment, the invention provides a method of administering an immunogenic composition comprising a S. aureus type 5 or 8 polysaccharide

covalently bound to carrier protein as described herein to a subject to generate an immune response as described herein.

[0062] In one embodiment, the invention provides a method of iso lating a polysaccharide with a molecular weight between 20 kDa and 1000 kDa.

[0063] In one embodiment, the invention provides an antibody generated by a capsular polysaccharide, an immunogenic conjugate, or an immunogenic composition of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

|0064] The present invention will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0065] Figu re 1 shows a repeating polysaccharide structure of S. aureus serotype 8 capsular polysaccharide (N-acetyl mannosaminuronic acid is ManNAca, N-acetyl L-fucosamine is L-FucNAc, and N-acetyl D-fucosamine is D-FucNAc).

[0066] Figure 2A shows an analysis of fractions from ion exchange

chromatography (Q-Sepharose) for S. aureus serotype 8 capsular polysaccharide (O-Acetyl Assay) and teichoic acid (phosphate assay); Figure 2B shows an analysis of fractions from ion exchange chromatography (Q-Sepharose) for S. aureus serotype 8 capsular polysaccharide by double immunodiffusion assay.

[0067] Figure 3A shows the effect of pH (3.5, 4 or 5) at 95°C on the reduction of S. aureus serotype 8 capsular polysaccharide molecular weight in heat treatment; Figure 3B shows the effect of temperature (55°C, 75°C or 95°C) at pH 3.5 on the reduction of S. aureus serotype 8 capsular polysaccharide molecular weight in heat treatment.

[0068] Figure 4 shows the molecular weight of purified 5. aureus serotype 8 capsular polysaccharide compared to serotype 5 capsular polysaccharide over time during heat treatment at pH 3.5 and 4.5, respectively, and 95°C.

[0069] Figure 5 shows increased survival in mice that received a serotype 8 capsular polysaccharide-CRM i 7 conjugate (diamonds) compared to ALP04-treated controls (circles).

[0070] Figure 6 shows a repeating polysaccharide structure of S. aureus serotype 5 polysaccharide ( V-acetyl mannosaminuronic acid is ManNAcA, iV-acetyl L-fucosamine is L-FucNAc, and /V-acetyl D-fucosamine is D-FucNAcA).

|0071 ] Figure 7A shows an analysis of fractions from ion exchange

chromatography (Q-Sepharose) for S. aureus serotype 5 polysaccharide (O-Acetyl Assay) and teichoic acid (phosphate assay); Figu re 7B shows an analysis of fractions from ion exchange chromatography (Q-Sepharose) for S. aureus serotype 5 polysaccharide by double immunodiffusion assay.

10072] Figure 8A shows the effect of pH (3.5, 4 or 5) at 95°C on the reduction of S. aureus serotype 5 capsular polysaccharide molecular weight in heat treatment; Figure 8B shows the effect of temperature (55°C, 75°C or 95°C) at pH 3.5 on the reduction of S. aureus serotype 5 capsular polysaccharide molecular weight in heat treatment.

J0073] Figure 9 shows reduced pyelonephritis in mice that received a serotype 5 polysaccharide-CRM i 97 conjugate compared to PBS-treated controls (shaded area is the treated mice).

|0074] Figure 10 shows colony forming units (CFU) recovered in kidneys after challenge with S. aureus PFESA0266 in mice vaccinated with high molecular weight (HMW) CP5-CRM, low molecular weight (LMW) CP5-CR or PP5-CR control

|0075] Figure 1 1 shows a comparison of OPA titers (geomean) from serum obtained from mice vaccinated with different formulations of polysaccharide conjugate (high molecular weight (HMW) CP5-CRM, low molecular weight (LMW) CP5-CRM). Groups consisted of 5 to 9 mice.

DETAI LED DESCRIPTION

Overview

|0076] The present invention relates to immunogenic conjugates comprising S. aureus serotype 5 or 8 capsular polysaccharides conjugated to carrier proteins and methods for their preparation and use. Novel features of the immunogenic conjugates of the invention include the molecular weight profiles of the polysaccharides and resulting conjugates, the ratio of conjugated lysines per CRM| 97 carrier protein and number of lysines covalently linked to the

polysaccharide, the number of covalent linkages between the carrier protein and the polysaccharide as a function of repeat units of the polysaccharide, and the relative amount of free polysaccharide compared to the total polysaccharide. The term "free polysaccharide" as used herein means a polysaccharide that is not conjugated to the carrier protein, but is nevertheless present in the conjugate composition.

|0077] Methods for making the immunogenic conjugates of the invention involve covalent conjugation of the capsular polysaccharides with the carrier proteins using conjugation chemistry involving CDI ( 1 , 1 -carbonyldiimidazole), CDT

( l , l -carboyl-di- l ,2,4-triazole) or PDPH (3-(2-pyridyldithio)-propionyl hydrazide). CDl is specific for CP8 conjugation only. Use of CDl/CDT results in a one-carbon or zero-carbon linker between capsular polysaccharide and carrier protein, while use of PDPH results in a covalent thioether bond between capsular polysaccharide and carrier protein.

|0078] Additional cross linkers for -SH (thiolated CP) to -NH2 linkages include but are not limited to: sulfo-LC-SM PT; sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamido]hexanoate)); sulfo-K US (N-[k-maleimidoundecanoyloxyjsulfosuccinimide ester); sulfo-LC-SPDP

(sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate) which cleaves by thiols; sulfo-SMPB (sulfosuccinimidyl 4-[/?-maleimidophenyl]butyrate); sulfo-SIAB (/V-sulfosuccinimidyl[4-iodoacetyl]aminobenzoate); sulfo-EMCS ([N-e-maleimidocaproyloxyjsulfosuccinimide ester); EMCA (N-e-maleimidocaproic acid); sulfo-SMCC (sulfosuccinimidyl 4-[ V-maleimidomethyl]cyclohexane- l -carboxylate); sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester); sulfo-GMBS (N-[g-maleimidobutyryloxy]sulfosuccinimide ester); BMPA (Ν-β-maleimidopropionic acid); 2-immunothiolane hydrochloride; 3-(2-pyridyldithio) propionic acid N-succinimidyl ester; 3-malemidopropionic acid N-succinimidyl ester; 4-maleimidobutyric acid N-succinimidyl ester; SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene); LC-SMCC

(succinimidyl-4-[N-maleimidomethyl]cyclohexane- l -carboxy-[6-amidocaproate]); K UA (N-k-maleimidoundecanoic acid); LC-SPDP (succinimidyl 6-(3-[2-

pyridyldithio]-propionamido)hexanoate); SMPH (succinimidyl-6-[P-maleimidopropionamido]hexanoate); SMPB (succinimidyl 4-[p-maleimidophenyljbutyrate); SIAB (N-succinimidyl[4-iodoacetyl]aminobenzoate); EMCS ([N-e-Maleimidocaproyloxy]succinimide ester); SMCC (succinimidyl 4-[iV-maleimidomethyl]cyclohexane- l -carboxylate); MBS (w-Maleimidobenzoyl-N-hydroxysuccinimide ester); SBAP (succinimidyl 3 -[bromoacetamido]propionate); BMPS (iV-[ -maleimidopropyloxy]succinimide ester); AMAS N-(a-maleimidoacetoxy) succinimide ester); SIA (Nsuccinimidyl iodoacetate); and N-succinimidyl (4-iodoacetyl)-aminobenzoate.

|0079] The agents can also be crosslinked using crosslinkers for -SH to -OH groups. Such cross linkers include but are not limited to PMPI (N-[/>-maleimidophenyl]isocyanate).

[0080] The compositions and methods described herein are useful in a variety of applicat ions. For example, the conjugates can be used in the production of conjugate immunogenic compositions to protect recipients from S. aureus infections. Alternatively, the various conjugates can be used in the production of antibodies against bacterial capsular polysaccharides, which subsequently can be used in research and clinical laboratory assays, such as bacterial detection and serotyping. Such antibodies may also be used to confer passive immunity to a subject. In some embodiments, the antibodies produced against bacterial polysaccharides are functional in either an animal efficacy model or in an opsonophagocytic killing assay.

|00811 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invent ion, the preferred methods and materials are described herein. In describing the embodiments and claiming the invention, certain terminology will be used in accordance with the definitions set out below.

|0082| As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, e.g. , references to "the method" includes one or more methods, and/or steps of the type described

herein and/or which will become apparent to one of ordinary skill in the art upon reading this disclosure and so forth.

[0083] As used herein, "about" means within a statistically meaningful range of a value such as a stated concentration range, time frame, molecular weight, temperature or pH. Such a range can be within an order of magnitude, typically within 20%, more typically still within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by the term "about" will depend upon the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Whenever a range is recited within this application, every whole number integer within the range is also contemplated as an embodiment of the invention.

We Claim:
1. A method of producing an immunogenic polysaccharide-protein conjugate comprising an isolated
Staphylococcus aureus serotype 8 capsular polysaccharide conjugated to a carrier protein, the method
comprising the steps of:
a) reacting an isolated S. aureus serotype 8 capsular polysaccharide with carbonyl ditriazole (CDT) in
an organic solvent to produce an activated serotype 8 polysaccharide; and
b) reacting the activated serotype 8 polysaccharide with a carrier protein in an organic solvent to
produce a serotype 8 polysaccharidexarrier protein conjugate;
wherein an immunogenic conjugate comprising a S. aureus serotype 8 capsular polysaccharide-carrier
protein conjugate is produced.
2. A method of producing an immunogenic polysaccharide-protein conjugate comprising an isolated
Staphylococcus aureus serotype 5 capsular polysaccharide conjugated to a carrier protein, the method
comprising the steps of:
a) reacting an isolated S. aureus serotype 5 capsular polysaccharide with carbonyl ditriazole (CDT) in
an organic solvent to produce an activated serotype 8 polysaccharide; and
b) reacting the activated serotype 5 polysaccharide with a carrier protein in an organic solvent to
produce a serotype 5 polysaccharidexarrier protein conjugate;
wherein an immunogenic conjugate comprising a S. aureus serotype 5 capsular polysaccharide-carrier
protein conjugate is produced.
3. The method of claim 1 or claim 2, which further comprises:
lyophilizing the isolated polysaccharide; and re-suspending the lyophilized polysaccharide in an organic
solvent.
4. The method of any one of claims 1-3 wherein the activated isolated polysaccharide is isolated from
the activation reaction prior to reacting with the carrier protein.
5. The method of claim 4, wherein step b) comprises:
i) lyophilizing the isolated activated isolated polysaccharide to produce a lyophilized activated isolated
polysaccharide;
ii) lyophilizing the carrier protein to produce a lyophilized carrier protein; and
iii) re-suspending the lyophilized activated isolated polysaccharide and the lyophilized carrier protein
in an organic solvent to produce an activated isolated polysaccharide mixed with a carrier protein.
6. The method of any of claims 1-5 , which further comprises dilution of the reaction mixture of step b)
into a buffer and maintaining a pH of about 8.8 to about 9.2 for at least 4 hours at about 20°C to about
26°C.
7. The method of any of claims 1-5, which further comprises dilution of the reaction mixture of step b)
into a buffer and maintaining a pH of about 9.0 for at least 4 hours at about 23°C.
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8. The method of any of claims 1-7, further comprising isolating the S. aureus serotype capsular
polysaccharide-carrier protein conjugate.
9. The method of any of claims 1-8, wherein the organic solvent is a polar aprotic solvent.
10. The method of claim 9, wherein the polar aprotic solvent is selected from the group consisting of
dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, V-methyl-2-pyrrolidone,
and hexamcthylphosphoramide (HMPA).
11. The method of claim 10, wherein the organic solvent is dimethyl sulfoxide (DMSO).
12. The method of any of claims 1 and 3-11 wherein the step of reacting the polysaccharide with CDT
comprises determining the water present in the S. aureus serotype 8 capsular polysaccharide and
adjusting the CDT concentration to about 1 : 1 molar ratio of CDT:water in the organic solvent.
13. The method of any of claims 1 and 3-12wherein the step of reacting the polysaccharide with CDT
comprises determining the water present in the S. aureus serotype 8 capsular polysaccharide and
adjusting the CDT concentration to about 0.5: 1 molar ratio of CDT:water in the organic solvent.
14. The method of any of claims 1 and 3-12 wherein the step of reacting the polysaccharide with CDT
comprises determining the water present in the S. aureus serotype capsular 8 polysaccharide and
adjusting the CDT concentration to about 0.75: 1 molar ratio of CDT:water in the organic solvent.
15. The method of any of claims 2-11, wherein the step of reacting the serotype 5 polysaccharide with
CDT comprises providing about 20-fold molar excess of CDT compared to the polysaccharide.
16. The method of any of claims 2-11 and 15 wherein the serotype 5 polysaccharide CDT in an organic
solvent mixture is adjusted to a water concentration between 0. 1 and 0.3%.
17. The method of claim 16 wherein the water concentration is adjusted to 0.2%.
18. The method of any of claims 3-17, wherein the step of isolating the activated serotype
polysaccharide comprises diafiltration.
19. The method of any of claims 1-18, wherein prior to lyophilization the carrier protein is diafiltered
against NaCl and the w/w ratio of NaCl/protein carrier protein is adjusted to about 0.5 to about 1.5.
20. The method of any of claims 1-19, wherein the carrier protein is CRM
21. The method of claim 20, wherein the activated serotype polysaccharide is reacted with the CRM197
in a ratio by weight of about 1:1.
22. The method of any of claims 1-21, wherein the isolated S. aureus serotype capsular polysaccharide
is mixed with imidazole or triazole prior to mixing with CDT in an organic solvent.
23. The method of any of claims 1-22 which further comprises hydrolyzing the serotype polysaccharidecarrier
protein conjugate to remove unreacted activation groups.
24. A method of making an immunogenic conjugate comprising an isolated Staphylococcus aureus
serotype 8 capsular polysaccharide conjugated to a carrier protein, the method comprising the steps
of:
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a) reacting a S. aureus serotype 8 capsular polysaccharide with 3-(2-pyridyldithio)-propionyl hydrazide
(PDPH) and a carbodiimide in an organic solvent to produce a PDPH-linked polysaccharide;
b) reacting the PDPH-linked polysaccharide with a reducing agent to produce an activated
polysaccharide;
c) isolating the activated serotype 8 polysaccharide to produce an isolated activated serotype 8
polysaccharide;
d) providing an activated carrier protein;
e) reacting the isolated activated serotype 8 polysaccharide with the activated carrier protein to
produce a serotype 8 polysaccharide-carrier protein conjugate; whereby an immunogenic conjugate
comprising an isolated S. aureus serotype 8 capsu lar polysaccharide conjugated to a carrier protein is
produced.
25. A method of making an immunogenic conjugate comprising an isolated Staphylococcus aureus
serotype 5 capsular polysaccharide conjugated to a carrier protein, the method comprising the steps
of:
a) reacting a S. aureus serotype 5 capsular polysaccharide with 3-(2-pyridyldithio)-propionyl hydrazide
(PDPH) and a carbodiimide in an organic solvent to produce a PDPH-linked polysaccharide;
b) reacting the PDPH-linked polysaccharide with a reducing agent to produce an activated
polysaccharide;
c) isolating the activated serotype 5 polysaccharide to produce an isolated activated serotype 5
polysaccharide;
d) providing an activated carrier protein:
e) reacting the isolated activated serotype 5 polysaccharide with the activated carrier protein to
produce a serotype 5 polysaccharide-carrier protein conjugate; whereby an immunogenic conjugate
comprising an isolated S. aureus serotype 5 capsu lar polysaccharide conjugated to a carrier protein is
produced.
26. The method of claim 24 or claim 25 wherein the activated carrier protein is isolated prior to
reacting the activated carrier protein with the activated polysaccharide.
27. The method of any one of claims 24-26, wherein:
step c) further comprises lyophilizing the isolated activated serotype 8 polysaccharide to
produce a lyophilized activated serotype polysaccharide.
28. The method of any of claims 24-27, wherein the organic solvent is a polar aprotic solvent.
29. The method of claim 28, wherein the polar aprotic solvent is selected from the group consisting of
dimethyl sulfoxide (DMSO),
dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, and
hexamethylphosphoramide (HMPA).
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30. The method of claim 55, wherein the polar aprotic solvent is dimethyl sulfoxide (DMSO).
31. The method of any of claims 24-30, wherein the carbodiimide is 1-Ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDAC).
32. The method of any of claims 24-31, wherein the step of reacting the serotype capsular
polysaccharide with PDPH and EDAC in an organic solvent comprises maintaining a polysaccharide:
PDPH : EDAC ratio by weight of about 1 :5 :3.
33. The method of any of claims 24-32, wherein the reducing agent is dithiothreitol (DTT).
34. The method of any of claims 24-33 wherein activation of the carrier protein comprises reacting the
carrier protein with a bromoacetic acid.
35 . The method of claim 34 wherein the bromoacetic acid comprises the N-hydroxysuccinimide ester
of bromoacetic acid (BAANS).
36. The method of any of claims 24-35, wherein the step of isolating the activated serotype
polysaccharide comprises diafiltration.
37. The method of any of claims 24-36 wherein the step of isolating the activated carrier protein
comprises diafiltration.
38. The method of any of claims 24-37 which further includes the step of hydrolyzing the serotype
polysaccharide-carrier protein conjugate to remove unreacted activation groups.
39. The method of claim 38 wherein the step of hydrolyzing the serotype polysaccharide-carrier protein
conjugate comprises the addition of cysteamine hydrochloride.
40. The method of any of claims 24-39 which further comprises isolating the immunogenic conjugate
comprising an isolated S. aureus serotype capsular polysaccharide conjugated to a carrier protein.
41. The method of claim 40 wherein the isolation of the serotype polysaccharidexarrier protein
conjugate comprises diafiltration.
42. The method of any of claims 24-41, wherein said carrier protein is CRM197.
43. The method of claim 42, wherein the activated serotype polysaccharide is reacted with the CRM in
a ratio by weight of about 1 : 1 .
44. The method of any of claims 1-43 wherein the activated polysaccharide has a size between about
20 kDa and about 1000 kDa.
45. The method of claim 44, wherein the activated polysaccharide has a size between about 50 kDa
and 500 kDa.
46. The method of any of claims 1-45 wherein the immunogenic polysaccharide-protein conjugate has a
size between 400 kDa and about 5000 kDa.
47. An immunogenic conjugate produced by the method of any of claims 1-46.
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48. The immunogenic conjugate of claim 47 which comprises more than about 1 % free polysaccharide
and less than about 30% free polysaccharide.
49. The immunogenic conjugate of claim 47 which comprises less than about 20% free polysaccharide.
50. An immunogenic composition comprising the immunogenic conjugate of any of claims 47-49 and at
least one of an adjuvant, diluent, or carrier.
51. The immunogenic composition of claim 50, wherein the adjuvant is an aluminum-based adjuvant.
52. The immunogenic composition of claim 51, wherein the adjuvant is selected from the group
consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
53. The immunogenic composition of claim 52 wherein the adjuvant is aluminum phosphate.
54. The immunogenic composition of any of claims 50-53 which comprises less than 30% free type
polysaccharide compared to the total amount of type polysaccharide.
55. The immunogenic composition of claim 54 which comprises less than 20% free type polysaccharide
compared to the total amount of type polysaccharide.
56. A S. aureus Type 8 capsular polysaccharide having a molecular weight of between 20 kDa and 1000
kDa covalently bound to a carrier protein; wherein the combined molecular weight of the
polysaccharide covalently bound to the carrier protein is between about 200 kDa and 5000 kDa.
57. A S. aureus Type 5 capsular polysaccharide having a molecular weight of between 20 kDa and 1000
kDa covalently bound to a carrier protein; wherein the combined molecular weight of the
polysaccharide covalently bound to the carrier protein is between about 200 kDa and 5000 kDa.
58. The polysaccharide covalently bound to carrier protein of claim 56 or claim 57 wherein the
polysaccharide has a molecular weight range of between 70 kDa and 300 kDa.
59. The polysaccharide covalently bound to carrier protein of claim 56 or claim 57 that has a molecular
weight range of between 500 kDa and 2500 kDa.
60. The polysaccharide covalently bound to carrier protein of claim 56 or claim 57, wherein the carrier
protein is CRM197.
61. The polysaccharide covalently bound to carrier protein of claim 60, wherein the CRM is covalently
linked to the polysaccharide through a carbamate linkage, an amide linkage, or both.
62. The polysaccharide covalently bound to carrier protein of claim 60 or claim 61, wherein the molar
ratio of conjugated lysines to CRM197 is about 10: 1 to about 25: 1.
63. The polysaccharide covalently bound to carrier protein of any one of claim 62, wherein at least one
covalent linkage between CRM197 and polysaccharide occurs at least at every 5 to 10 saccharide repeat
units of the polysaccharide.
64. The polysaccharide covalently bound to earner protein of claim 62 or claim 63, wherein at least
one linkage between CRM197 and polysaccharide occurs at every 5 saccharide repeat units of the
polysaccharide.
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65. The polysaccharide covalently bound to carrier protein of any of claims 62-63 wherein the CRM197
comprises 5 to 22 lysines covalently linked to the polysaccharide.
65. The polysaccharide covalently bound to carrier protein of any of claims 62-63 wherein the CRM197
comprises 5 to 22 lysines covalently linked to the polysaccharide.
66. The polysaccharide covalently bound to carrier protein of claim 65, wherein the CRM197 comprises 8
to 15 lysines covalently linked to the polysaccharide.
67. An immunogenic composition comprising the polysaccharide covalently bound to carrier protein of
any of claims 56-66 and at least one of an adjuvant, diluent, or carrier.
68. The immunogenic composition of claim 67, wherein the adjuvant is an aluminum-based adjuvant.
69. The immunogenic composition of claim 68, wherein the adjuvant is selected from the group
consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
70. The immunogenic composition of claim 69, wherein the adjuvant is aluminum phosphate.