巴西专利BR112012019757B1 IMMUNOGENIC COMPOSITION, AND, USE OF A MULTI VALENT POLYSACARIDE-PROTEIN CONJUGATE MIXTURE

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IMMUNOGENIC COMPOSITION, AND, USE OF A MULTIVALENT POLYSACARID-PROTEIN CONJUGATE MIXTURE The present invention provides a multivalent immunogenic composition having 15 distinct polysaccharide-protein conjugates. Each conjugate consists of a capsular polysaccharide prepared from 14.18C, 19A, 19F, 22F, 23F or 33F) conjugated to a carrier protein, preferably CRM197. The immunogenic composition, preferably formulated as a vaccine in an aluminum-based adjuvant, provides broad coverage against pneumococcal disease, particularly in babies and young children.
公开号:BR112012019757B1
申请号:R112012019757-6
申请日:2011-02-03
公开日:2020-11-17
发明作者:Michael J. Caulfield；Patrick L. Ahl；Jeffrey T. Blue；Jayme L. Cannon
申请人:Merck Sharp & Dohme Corp；
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CROSS REFERENCE TO RELATED REQUESTS
None FIELD OF THE INVENTION
The present invention provides a multivalent immunogenic composition having 15 distinct polysaccharide-protein conjugates. Each conjugate consists of a capsular polysaccharide prepared from a different serotype of Streptococcus pneumoniae (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F or 33F) conjugated to a carrier protein, preferably CRMI97. The immunogenic composition, preferably formulated as a vaccine in an aluminum-based adjuvant, provides broad coverage against pneumococcal disease, particularly in babies and young children. BACKGROUND OF THE INVENTION
Streptococcus pneumoniae is a significant cause of serious illness worldwide. In 1997, the Centers for Disease Control and Prevention (CDC) estimated that there were 3,000 cases of pneumococcal meningitis, 50,000 cases of pneumococcal bacteremia, 7,000,000 cases of pneumococcal otitis media and 500,000 cases of pneumococcal pneumonia annually in the United States. See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997, 46 (RR-8): 1-13. In addition, complications from these diseases can be significant with some studies reporting up to 8% mortality and 25% neurological sequelae with pneumococcal meningitis. See Arditi etal., 1998, Pediatrics 102: 1087-97.
Multivalent pneumococcal polysaccharide vaccines that have been licensed for many years have been shown to be valuable in preventing pneumococcal disease in adults, particularly the elderly and those at high risk. However, babies and young children respond insufficiently to unconjugated pneumococcal polysaccharides. The pneumococcal conjugate vaccine, Prevnar®, containing the 7 most frequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) that currently causes invasive pneumococcal disease in young children and babies, was first licensed in the United States in February 2000. Following the universal use of Prevnar® in the United States, there was a significant reduction in invasive pneumococcal disease in children due to the serotypes present in Prevnar®. See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2005, 54 (36): 893-7. However, there are limitations on serotype coverage with Prevnar® in certain regions of the world and some evidence for certain emerging serotypes in the United States (for example, 19A and others). See O'Brien et al., 2004, Am J Epidemiol 159: 634-44; Whitney et al., 2003, N Engl J Med 348: 1737-46; Kyaw et al., 2006, N Engl J Med 354: 1455-63; Hicks et al., 2007, J Infect Dis 196: 1346-54; Traore et al., 2009, Clin Infect Dis 48: S181- S189.
US Patent Application Publication 2006/0228380 A1 describes a vaccine with 13-valent pneumococcal polysaccharide-protein conjugate including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Chinese Patent Application Publication No. CN 101590224 A describes a vaccine with 14-valent pneumococcal polysaccharide-protein conjugate including serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19Fe23F.
Other PCVs have covered 7, 10, 11, or 13 of the serotypes contained in PCV-15, but immune interference has been observed for some serotypes (for example, lower protection for serotype 3 in PCV-11 from GSK) and rates of lower response rates for serotype 6B on Pfizer PCV-13. See Prymula et al., 2006, Lancet 367: 740-48 and Kieninger et al., Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers, presented at the 48th Annual ICAAC / 460 ISDA Annual Meeting, Washington DC, October 25-28, 2008. SUMMARY OF THE INVENTION
The present invention provides an immunogenic composition comprising (1) a multivalent polysaccharide-protein conjugate consisting of capsular polysaccharides from 15 different S. pneumonia serotypes coupled to a carrier protein, and (2) a pharmaceutically acceptable carrier. More specifically, the present invention provides a vaccine composition with 15-valent pneumococcal conjugate (PCV-15) that comprises a multivalent polysaccharide-protein conjugate consisting of capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F of S. pneumonia attached to a carrier protein; and a pharmaceutically acceptable carrier. In a specific embodiment, the immunogenic composition contains capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F and the carrier protein is CRM197 .
In certain embodiments, the composition further comprises an adjuvant. In certain embodiments, the adjuvant is an aluminum based adjuvant, such as aluminum phosphate, aluminum sulfate or aluminum hydroxide. In a particular embodiment of the invention, the adjuvant is aluminum phosphate.
The present invention also provides a method for inducing an immune response to a S. pneumoniae capsular polysaccharide, which comprises administering to a human an immunologically effective amount of the above immunogenic composition.
The present invention further provides an immunogenic composition administered as a single 0.5 ml dose formulated to contain: 2 pg of each polysaccharide, except for 6B to 4 pg; about 32 pg of CRMI97 carrier protein; 0.125 mg of elemental aluminum adjuvant (0.5 mg of aluminum phosphate); 150 mM sodium chloride and 20 mM L-histidine buffer. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Comparison of GMCs for PCV-15 relative to Prevnar® in Baby Rhesus Monkeys (Prevnar serotypes, PD-2 and PD-3). The error bars indicate 2 standard errors.
Figure 2: Serotype specific GMCs for non-Prevnar® serotypes in Baby Rhesus Monkeys immunized with PCV-15. The error bars indicate 2 standard errors.
Figure 3: NZWR-1: Comparison of geometric mean titers in rabbits immunized with PCV-15 without (OxA) or with APA (81) (Post dose 2). The error bars indicate 95% of Cl in the difference of times in the geometric mean (PCV-15 without APA / PCV-15 with APA). DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a multivalent immunogenic composition comprising, consisting essentially of, or alternatively, consisting of 15 distinct polysaccharide-protein conjugates, each conjugate containing a different capsular polysaccharide conjugated to a carrier protein, and in which capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 68, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae, together with a pharmaceutically acceptable carrier. In certain embodiments, the carrier protein is CRMI97. The immunogenic composition can further comprise an adjuvant, such as an aluminum based adjuvant, such as aluminum phosphate, aluminum sulfate and aluminum hydroxide. The present invention also provides a method for inducing an immune response to a S. pneumoniae conjugated capsular polysaccharide, which comprises administering to a human an immunologically effective amount of the above multivalent immunogenic composition.
As illustrated in the Examples, below, preclinical studies in baby Rhesus monkeys have demonstrated robust antibody responses to all 15 serotypes in PCV-15 that are comparable to the responses to the 7 common serotypes in Prevnar®. Applicants' finding that a vaccine with a 15-valent pneumococcal conjugate including the addition of new polysaccharide-protein conjugates containing 22F and 33F serotypes provides robust antibody responses demonstrates the feasibility of expanding the coverage of pneumococcal serotypes not covered by existing pneumococcal vaccines.
The term "comprises" when used with the immunogenic composition of the invention refers to the inclusion of any of the other components (subject to the limitations of the language "consisting of" for the antigen mixture), such as adjuvants and excipients. The term "consisting of 'when used with the multivalent polysaccharide-protein conjugate mixture refers to a mixture having these 15 S' polysaccharide protein conjugates, pneumoniaeparticulars and no other of the S. pneumoniae protein polysaccharide conjugates one different serotype. Capsular polysaccharide-protein conjugates of Streptococcus pneumoniae
Steptococcus pneumoniae capsular polysaccharides can be prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be isolated from bacteria and can be adjusted to some degree by known methods (see, for example, European Patents EP497524 and EP497525) and preferably by microfluidization. Polysaccharides can be adjusted to reduce viscosity in polysaccharide samples and / or to improve filterability for conjugated products. In the present invention, capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae.
In one embodiment, each pneumococcal polysaccharide serotype is grown in a soy-based medium. The individual polysaccharides are then purified through standard steps including centrifugation, precipitation, and ultra-filtration. See, for example, US Patent Application Publication 2008/0286838 and Pat. US 5,847,112.
Carrier proteins are preferably proteins that are non-toxic and non-reactogenic and obtainable in sufficient quantity and purity. A carrier protein can be conjugated or joined with an S. pneumoniae polysaccharide to enhance the immunogenicity of the polysaccharide. Carrier proteins must be subject to standard conjugation procedures. In a particular embodiment of the present invention, CRMI97 is used as the carrier protein. In one embodiment, each capsular polysaccharide is conjugated to the same carrier protein (each capsular polysaccharide molecule that is conjugated to a single carrier protein). In another embodiment, the capsular polysaccharides are conjugated to two or more carrier proteins (each capsular polysaccharide molecule being conjugated to a single carrier protein). In such an embodiment, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein.
CRM197 is a non-toxic (ie, toxoid) variant of diphtheria toxin. In one embodiment, it is isolated from Corynebacterium diphtheriacepa C7 ((3197) cultures grown on casamino acids and medium based on yeast extract. In another embodiment, CRM197 is prepared recombinantly according to the methods described in US 5,614,382. Typically, CRM497 is purified through a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. In some embodiments, CRM197 is prepared in Pseudomonas fluorescens using Pfenex Expression Technology® (Pfenex Inc., San Diego, CA).
Other suitable carrier proteins include additional inactivated bacterial toxins such as DT (diphtheria toxoid), TT (tetanus toxoid) or C fragment of TT, pertussis toxoid, cholera toxoid (for example, as described in the International Patent Application Publication No. WO 2004/083251), E. coliLT, E. coli ST, and Pseudomonas aeruginosa exotoxin A. Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding proteins, protein A pneumococcal surface (PspA; See International Patent Application Publication No. WO 02/091998), pneumococcal adhesin protein (PsaA), Group A or Group B streptococcal peptidase C5a, or Haemophilus influenzae protein, pneumococcal pneumolysin ( Kuo et al., 1995, Infect Immun 63; 2706-13) including detoxified ply in some way for example dPLY-GMBS (See International Patent Application Publication No. WO 04/081515) or dPLY-formaldehyde, PhtX, included including PhtA, PhtB, PhtD, PhtE and Pht protein fusions for example PhtDE fusions, PhtBE fusions (See International Patent Application Publications No. WO 01/98334 and WO 03/54007), can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein derivative (PPD), PorB (from N. meningitidis), PD (protein D from Haemophilus influenzae- , see, for example, European Patent No. EP 0 594 610 B), or equivalents of these immunologically functional, synthetic peptides (See European Patent Nos. ~ EP0378881 and EP0427347), heat shock proteins (See International Patent Application Publications No. ~ WO 93/17712 and WO 94/03208), pertussis proteins (See International Patent Application Publication No. WO 98/58668 and European Patent No. EP0471177), cytokines, lymphokines, growth factors or hormones (See Publication of International Patent Application No. WO 91/01146), artificial proteins comprising multiple human CD4 + T cell epitopes from various pathogen-derived antigens (See Falugi et al., 2001, Eur J Immunol 31: 3816-3824) such as protein N19 (V er Baraldoi et al., 2004, Infect Immun 72: 4884-7), iron uptake proteins (See International Patent Application Publication WO 01/72337), C. difficile toxin A or B (See Publication International Patent No. WO 00/61761), and flagellin (See Ben-Yedidia et al., 1998, Immunol Left 64: 9) can also be used as carrier proteins.
Other DT mutants can be used, such as CRM 176, CRM228, CRM 45 (Uchida et al., 1973, T Biol Chem 218: 3838-3844); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 for Asp, Gin or Ser and / or Ala 158 for Gly and other mutations disclosed in Pat. US 4,709,017 or Pat. US 4,950,740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations disclosed in U.S. Pat. US 5,917,017 or Pat. US 6,455,673; or fragment disclosed in U.S. Pat. US 5,843,711.
The purified polysaccharides are chemically activated to manufacture the saccharides capable of reacting with the carrier protein. Once activated, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. Polysaccharide conjugates can be prepared by known ligation techniques.
In one embodiment, the chemical activation of the polysaccharides and the subsequent conjugation to the carrier protein are obtained by means described in Pat. US 4,365,170, 4,673,574 and 4,902,506. In summary, this chemistry necessarily leads to the activation of pneumococcal polysaccharide by reaction with any oxidizing agent that oxidizes a terminal hydroxyl group to an aldehyde, such as periodate (including sodium periodate, potassium periodate, or periodic acid). The reaction leads to a random oxidative cleavage of vicinal hydroxyl groups of carbohydrates with the formation of reactive aldehyde groups.
Binding to the carrier protein (eg, CRM197) can be by reductive amination via direct amination to the protein's lysyl groups. For example, conjugation is accomplished by reacting a mixture of the activated polysaccharide and the carrier protein with a reducing agent such as sodium cyanoborohydride. The unreacted aldehydes are then capped with the addition of a strong reducing agent, such as sodium borohydride.
In another embodiment, the conjugation method may rely on the activation of the saccharide with l-cyan-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide can thus be linked directly or via a spacer group (linker) to an amino group on the carrier protein. For example, the spacer can be cystamine or cysteamine to give a thiolated polysaccharide that can be attached to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (for example using GMBS) or a haloacetylated carrier protein (eg example using iodoacetimide [e.g. ethyl iodoacetimide HCI] or N-succinimidyl bromoacetate or STAB, or STA, or SBAP). Preferably, the cyanate ester (optionally manufactured by CDAP chemistry) is bonded with hexane diamine or adipic acid dihydrazide (ADH) and the amino-derived saccharide is conjugated to the carrier protein using carbodiimide chemistry (for example EDAC or EDC) via a carboxyl group on the protein carrier. Such conjugates are described in International Patent Application Publications No. WO 93/15760, WO 95/08348 and WO 96/29094; and Chu et al., 1983, Infect. Immunity 40: 245-256.
Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in International Patent Application Publication No. WO 98/42721. The conjugation may involve a carbonyl ligand that can be formed by reacting a free hydroxyl group of the saccharide with CDI (See Betell et al., 1979, J. Biol. Chem. 254: 2572-4; Heam et al., 1981 , J. Chromatogr. 218: 509-18) followed by reaction with a protein to form a carbamate bond. This may involve reducing the anomeric terminal to a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a carbamate CDI intermediate and linking the carbamate CDI intermediate with an amino group in a protein.
In one embodiment, prior to formulation, each pneumococcal capsular polysaccharide antigen is individually purified from S. pneumoniae, activated to form reactive aldehydes, and then covalently conjugated using reductive amination to the CRM197 carrier protein.
After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugates are purified (enriched with respect to the amount of polysaccharide-protein conjugate) by one or more of a variety of techniques. Examples of these techniques are well known to the skilled person and include concentration / diafiltration, ultrafiltration, precipitation / elution, column chromatography, and deep filtration operations. See, for example, Pat. US 6,146,902. Pharmaceutical Compositions / Vaccine
The present invention further provides compositions, including pharmaceutical, immunogenic and vaccine compositions, which comprise, which consist essentially of, or alternatively, which consist of 15 distinct polysaccharide-protein conjugates, wherein each conjugate contains a different conjugated capsular polysaccharide to a carrier protein, and in which capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae , together with a pharmaceutically acceptable carrier and adjuvant. These pneumococcal conjugates are prepared by separate processes and formulated in bulk in a single dosage formulation.
As defined herein, an "adjuvant" is a substance that serves to enhance the immunogenicity of an immunogenic composition of the invention. An immune adjuvant can enhance an immune response to an antigen that is weakly immunogenic when administered alone, for example, inducing none or weak antibody titers or cell-mediated immune response, increases antibody titers to the antigen, and / or decreases the dose of the antigen effective to obtain an immune response in the individual. Thus, adjuvants are often administered to enhance the immune response and are well known to the skilled person. Suitable adjuvants to enhance the effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc .; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (defined below) or bacterial cell wall components), such as, for example, (a) MF59 (Patent Application Publication International No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as the Model HOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked L121 polymer, and microfluidized thr-MDP in a submicron or eddy to generate a large particle size emulsion, (c) Ribi® adjuvant system (RAS), (Corixa, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more components of bacterial cell wall of the group consisting of monophosphorylipid A (MPL®) 3-O-delaminated described in U.S. Pat. US 4,912,094, trehalose dimicolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox®); and (d) a Montanide ISA; (3) saponin adjuvants, such as Quil A or STIMULON® QS-21 (Antigenics, Framingham, MA) (see, for example, US Pat. 5,057,540) can be used or particles generated from them such as ISCOM (immunostimulatory complexes formed by the combination of cholesterol, saponin, phospholipid, and antipathetic proteins) and Iscomatrix (having essentially the same structure as an ISCOM but without the protein); (4) bacterial lipopolysaccharides, synthetic lipid A analogues such as aminoalkyl glucosamine phosphate (AGP) compounds, or derivatives or analogues thereof, which are available from Corixa, and which are described in US Patent 6,113,918; such an AGP is 2 - [(R) -3-tetra-decanoyloxytetradecanoylamino] ethyl 2-Deoxy-4-O-phosphono-3-O - [(R) -3-tetradecanoyloxytetradecanoyl] -2- [(R) -3 -tetradecanoyloxytetradecanoyl-amino] - bD-glycopyranoside, which is also known as 529 (formerly known as RC529), which is formulated as an aqueous form or as a stable emulsion (5) synthetic polynucleotides such as oligonucleotides containing CpG ((s) motif) US Pat. 6,207,646); and (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc. .), interferons (for example, gamma interferon), macrophage granulocyte colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), B7- 1 and B7-2, etc; and (7) complement, such as a complement component trimer Cad.
In another embodiment, the adjuvant is a mixture of 2, 3, or more of the above adjuvants, for example, SBAS2 (an oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).
Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2- (r-2'-dipalmitoyl- sn-glycero-3-hydroxy-phosphoryloxy) -ethylamine (MTP-PE), etc.
In certain embodiments, the adjuvant is an aluminum salt. The aluminum salt adjuvant can be an alum precipitated vaccine or an alum absorbed vaccine. Aluminum salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts Research in Immunology 143: 489-493). The aluminum salt includes, but is not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, Amphogel, aluminum (III) hydroxide , hydroxyphosphate aluminum sulfate (Aluminum phosphate adjuvant (APA)), amorphous alumina, alumina trihydrate, or trihydroxyaluminium.
APA is an aqueous suspension of aluminum hydroxyphosphate. APA is manufactured by mixing aluminum chloride and sodium phosphate in a volumetric ratio of 1: 1 to precipitate aluminum hydroxyphosphate. After the mixing process, the material is reduced in size with a high shear mixer to obtain a particular target aggregate size in the range of 2 to 8 pm. The product is then diafiltered against physiological saline and sterilized with steam.
In certain embodiments, a commercially available A1 (OH) 3 (for example Alhydrogel or Superfos from Denmark / Accurate Chemical and Scientific Co., Westbury, NY) is used to absorb proteins in a ratio of 50 to 200 g of protein / mg of aluminum hydroxide. Protein absorption is dependent, in another embodiment, on the pi (Isoelectric pH) of the protein and the pH of the medium. A protein with a lower pi absorbs for the positively charged aluminum ion more strongly than a protein with a higher PI. Aluminum salts can establish a deposit of Ag that is released slowly over a period of 2 to 3 weeks, be involved in nonspecific activation of macrophages and activation of complement, and / or stimulate the innate immune mechanism (possibly through the stimulation of uric acid). See, for example, Lambrecht et al., 2009, Curr Opin Immunol 21: 23.
Monovalent aqueous bulk conjugates are typically mixed with each other and diluted to target 8 µg / ml for all serotypes except 6B, which will be diluted to target 16 µg / ml. Once diluted, the batch will be sterilized by filtration, and an equal volume of aluminum phosphate adjuvant added aseptically to target a final aluminum concentration of 250 pg / ml. The batch with adjuvant, formulated, will be filled in single use vials at 0.5 ml / dose.
In certain embodiments, the adjuvant is a nucleotide sequence containing CpG, for example, an oligonucleotide containing CpG, in particular an oligodeoxy-nucleotide containing CpG (CpG ODN). In another embodiment, the adjuvant is ODN 1826, which can be purchased from Coley Pharmaceutical Group.
The "CpG-containing nucleotide," "CpG-containing oligonucleotide," "CpG oligonucleotide," and similar terms refer to a nucleotide molecule of 6 to 50 nucleotides in length that contains an unmethylated CpG portion. See, for example, Wang et al., 2003, Vaccine 21: 4297. In another embodiment, any other definition accepted in the technique of terms is intended. Oligonucleotides containing CpG include modified oligonucleotides using any of the synthetic intemucleoside bonds, modified base and / or modified sugar.
Methods for using CpG oligonucleotides are well known in the art and are described, for example, in Sur et al., 1999, J Immunol. 162: 6284-93; Verthelyi, 2006, Methods Mol Med. 127: 139-58; and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst. 23: 89-110. Administration / Dosage
The compositions and formulations of the present invention can be used to protect or treat a human subject susceptible to pneumococcal infection, by administering the vaccine through a systemic or mucosal route. In one embodiment, the present invention provides a method for inducing an immune response to a S. pneumoniae conjugated capsular polysaccharide, which comprises administering to a human an immunologically effective amount of an immunogenic composition of the present invention. In another embodiment, the present invention provides a method of vaccinating a human against a pneumococcal infection, which comprises the step of administering to the human an immunologically effective amount of an immunogenic composition of the present invention.
"Effective amount" of a composition of the invention refers to a dose required to evoke antibodies that significantly reduce the likelihood or severity of pneumonia infectivity during a subsequent inoculation.
The methods of the invention can be used for the prevention and / or reduction of primary clinical syndromes caused by S. pneumonia including both invasive infections (meningitis, pneumonia, and bacteremia), as well as non-invasive infections (acute otitis media, and sinusitis).
Administration of the compositions of the invention may include one or more of: injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or through mucosal administration for oral / alimentary, respiratory or genitourinary tracts. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (since nasopharyngeal pneumococcal transport can be more effectively prevented, thus lessening the infection in its earliest stage).
The amount of conjugate in each dose of vaccine is selected as an amount that induces an immunopotective response without significant adverse effects. Such amount may vary depending on the pneumococcal serotype. In general, each dose will comprise from 0.1 to 100 pg of each polysaccharide, particularly from 0.1 to 10 pg, and more particularly from 1 to 5 pg. For example, each dose can comprise 100, 150, 200, 250, 300, 400, 500, or 750 ng or 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9 , 10, 11, 12, 13,14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, or 100 pg.
The ideal amounts of components for a particular vaccine can be ascertained by standard studies involving the observation of appropriate immune responses in individuals. For example, in another embodiment, the dosage for human vaccination is determined by extrapolating from animal studies to human data. In another embodiment, the dosage is determined empirically.
In one embodiment, the aluminum salt dose is 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500, or 700 pg, or 1, 1.2 , 1.5, 2, 3, 5 mg or more. In yet another embodiment, the dose of alum salt described above is per pg of recombinant protein.
In a particular embodiment of the present invention, the PCV-15 vaccine is a sterile liquid formulation of pneumococcal capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F , 22F, 23F and 33F individually conjugated to CRMI97. Each 0.5 ml dose is formulated to contain: 2 pg of each saccharide, except for 6B to 4 pg; about 32 pg of CRM197 carrier protein (for example, 32 pg ± 5 pg, ± 3 pg, ± 2 pg, or ± 1 pg); 0.125 mg of elemental aluminum adjuvant (0.5 mg of aluminum phosphate); and sodium chloride and L-histidine buffer. The sodium chloride concentration is about 150 mM (for example, 150 mM ± 25 mM, 120 mM, ± 15 mM, ± 10 mM, or ± 5 mM) and about 20 mM (for example, 20 mM ± 5 mM, 2.5 mM, ± 2 mM, ± 1 mM, or ± 0.5 mM) of L-histidine buffer.
According to any of the methods of the present invention and in an embodiment, the individual is the human being. In certain embodiments, the human patient is a baby (less than 1 year old), child (approximately 12 to 24 months), or young child (approximately 2 to 5 years). In other embodiments, the human patient is an elderly patient (> 65 years). The compositions of this invention are also suitable for use with older children, adolescents and adults (for example, at the age of 18 to 45 years or from 18 to 65 years).
In an embodiment of the methods of the present invention, a composition of the present invention is administered as a single inoculation. In another embodiment, the vaccine is administered twice, three times or four times or more, adequately spaced. For example, the composition can be administered at intervals of 1,2, 3, 4, 5, or 6 months or any combination thereof. The immunization program can follow that designated for pneumococcal vaccines. For example, the routine program for babies and children against the invasive disease caused by S ', pneumoniae is 2, 4, 6 and 12 to 15 months old. Thus, in a preferred embodiment, the composition is administered as a series of 4 doses at 2, 4, 6, and 12 to 15 months of age.
The compositions of this invention may also include one or more S. pneumoniae proteins. Examples of S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publications No. WO 02/083855 and WO 02/053761. Formulations
The compositions of the invention can be administered to an individual by one or more methods known to a person skilled in the art, such as parenteral, transmucosal, transdermal, intramuscular, intravenous, intradermal, intranasal, subcutaneous, intraperitoneal, and formulated accordingly.
In one embodiment, the compositions of the present invention are administered via epidermal injection, intramuscular injection, intravenous, intraarterial, subcutaneous injection, or intra-respiratory mucosal injection of a liquid preparation. Liquid formulations for injection include, solutions and the like.
The composition of the invention can be formulated as single dose vials, multiple dose vials or as pre-filled syringes.
In another embodiment, the compositions of the present invention are administered orally, and are thus formulated in a form suitable for oral administration, that is, as a solid or a liquid preparation. Solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soy oil, olive oil, sunflower oil, fish liver oil, another marine oil, or a milk lipid or eggs.
The pharmaceutical composition can be isotonic, hypotonic or hypertonic. However, it is often preferred that a pharmaceutical composition for infusion or injection is essentially isotonic when it is administered. Consequently, for storage the pharmaceutical composition can preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it can be diluted to make an isotonic solution before administration.
The isotonic agent can be an ionic isotonic agent such as a salt or a nonionic isotonic agent such as a carbohydrate. Examples of ionic isotonic agents include but are not limited to NaCl, CaCl2, KC1 and MgCl2. Examples of non-ionic isotonic agents include but are not limited to mannitol, sorbitol and glycerol.
It is also preferred that at least one pharmaceutically acceptable additive is a buffer. For some purposes, for example, when the pharmaceutical composition is intended for infusion or injection, it is often desirable for the composition to comprise a buffer, which is capable of buffering a solution at a pH in the range of 4 to 10, such as 5 to 9, for example 6 to 8.
The buffer for example can be selected from the group consisting of TRIS buffer, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine.
The buffer furthermore for example can be selected from USP compatible buffers for parenteral use, in particular, when the pharmaceutical formulation is for parenteral use. For example the buffer can be selected from the group consisting of monobasic acids such as acetic, benzoic, glyconic, glycolic and lactic; dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric, polybasic acids such as citric and phosphoric; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.
Parenteral vehicles (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, lactated Ringer's dextrose and sodium chloride and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, glycols such as propylene glycol or polyethylene glycol, and Polysorbate-80 are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soy oil, olive oil, sunflower oil, fish liver oil, another marine oil, or a milk lipid or eggs.
The formulations of the invention can also contain a surfactant. Preferred surfactants include, but are not limited to: polyoxyethylene sorbitan esters surfactants (commonly referred to as Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the brand name DOWFAX®, such as linear BO / PO block copolymers; octoxynols, which may vary in the number of repeating ethoxy groups (1,2-ethoxy-oxy), with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol® NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. A preferred surfactant to include in the emulsion is Tween 80 (polyoxyethylene sorbitan monooleate).
Mixtures of surfactants can be used, for example Tween 80 / Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) it is also suitable. Another useful combination comprises lauret 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.
Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as Tween 80) from 0.01 to 1%, in particular from about 0.1%; octyl- or nonylphenoxy-polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) from 0.001 to 0.1%, in particular from 0.005 to 0.02%; polyoxyethylene ethers (such as lauret 9) from 0.1 to 20%, preferably from 0.1 to 10% and in particular from 0.1 to 1% or from about 0.5%.
In another embodiment, the pharmaceutical composition is released in a controlled release system. For example, the agent can be administered using intravenous infusion, a transdermal patch, liposomes, or other modes of administration. In another embodiment, polymeric materials are used; for example in microspheres or an implant.
Also comprised by the invention are compounds modified by the covalent bonding of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Such modifications can increase the solubility of the compound in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the reactogenicity of the compound. In another embodiment, the desired in vivo biological activity is obtained by administering such polymer-compound adducts less frequently or in lower doses than with the unmodified compound.
In a preferred embodiment, the vaccine composition is formulated in L-histidine buffer with sodium chloride.
Having described the various embodiments of the invention with reference to the description and accompanying drawings, it should be understood that the invention is not limited to these precise embodiments, and that various changes and modifications can be made at that point by a person skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.
The following examples illustrate, but do not limit the invention. EXAMPLES EXAMPLE 1: Preparation of Capsular Polysaccharides X Pneumoniae
Methods of growing pneumococci are well known in the art. See, for example, Chase, 1967, Methods of Immunology and Immunochemistry 1: 52. Methods of preparing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European Patent No. EPO497524. Isolates of pneumococcal subtypes are available from the ATCC.
The bacteria are identified as encapsulated, non-mobile, Gram positive, lancet-shaped diplococci that are alpha-hemolytic in blood-agar. Subtypes are differentiated based on the Quelling reaction using specific antisera. See, for example, Pat. US 5,847,112. Cell Banks
The cell banks that represent each of the S. pneumococcus serotypes present in PCV-15 were obtained from the Merck Culture Collection (Rahway, NJ) in a frozen bottle. Inoculation
A thawed seed culture was transferred to the seed fermenter containing an appropriate pre-sterilized growth medium. Seed Fermentation
The culture was grown in the seed fermenter with temperature and pH control. The entire volume of the seed fermenter was transferred to the production fermenter containing pre-sterilized culture medium. Production Fermentation
Production fermentation was the final cell growth stage of the process. Temperature, pH and agitation rate were controlled. Inactivation
The fermentation process was terminated by adding an inactivating agent. After inactivation, the batch was transferred to the inactivation tank where it was kept at controlled temperature and agitation. Purification
The cell fragments were removed using a combination of centrifugation and filtration. The batch was ultrafiltered and diafiltered. The batch was then individualized for fractionation based on solvent that remove impurities and recover the polysaccharide. EXAMPLE 2: Preparation of Pneumococcal Polysaccharide-CRMiov Conjugates Activation Process
The saccharides of different serotype are individually conjugated to the purified CRM197 carrier protein using a common process flow. In this process the saccharide is dissolved, adjusted to a target molecular mass, chemically activated and exchanged in buffer for ultrafiltration. The purified CRM197 is then conjugated to the activated saccharide and the resulting conjugate is purified by ultrafiltration before final 0.2 µm membrane filtration. Various process parameters within each step, such as pH, temperature, concentration, and time, are serotype specific as described in this example. Step 1: Dissolution
The purified polysaccharide was dissolved in water at a concentration of 2 to 3 mg / ml. The dissolved polysaccharide was passed through a mechanical homogenizer with preset pressure from 0 to 1000 bar. Following the reduction in size, the saccharide was concentrated and diafiltered with sterile water in a 10 kDa MWCO ultrafilter. The permeate was discarded and the retentate was adjusted to a pH of 4.1 with a sodium acetate buffer, 50 mM final concentration. For serotypes 4 and 5, 100 mM sodium acetate at pH 5.0 was used. For serotype 4, the solution was incubated at 50 ° ± 2 ° C. Hydrolysis was stopped by cooling to 20 ° to 24 ° C. Step 2: Periodate reaction
The molar equivalents of sodium periodate required for the activation of pneumococcal saccharide were determined using the total saccharide content. With complete mixing, the oxidation was allowed to process between 3 and 20 hours at 20 to 24 ° C for all serotypes except 5, 7F, and 19F for which the temperature was 2 to 6 ° C. Step 3: Ultrafiltration
The oxidized saccharide was concentrated and diafiltered with 10 mM potassium phosphate, pH 6.4 (10 mM sodium acetate, pH 4.3 for serotype 5) in a 10 kDa MWCO ultrafilter. The permeate was discarded and the retained was adjusted to a pH of 6.3 to 8.4 by the addition of 3 M potassium phosphate buffer. Conjugation Process Step 1: Conjugation Reaction
The concentrated saccharide was mixed with CRM497 carrier protein at a loading ratio of 0.2 to 2 to 1. The mixed saccharide-CRMi97 mixture was filtered through a 0.2 pm filter.
The conjugation reaction was initiated by the addition of a solution of sodium cyanoborohydride to obtain 1.8 to 2.0 moles of sodium cyanoborohydride per mole of saccharide. The reaction mixture was incubated for 48 to 120 hours at 20 to 24 ° C (8 to 12 ° C for serotypes 3, 5, 6A, 7F, 19A, and 19F). Step 2: Reaction of Borohydride
At the end of the conjugation incubation, the reaction mixture was adjusted from 4 to 8o C, and a pH of 8 to 10 with 1.2 M sodium bicarbonate buffer or 3 M potassium phosphate buffer (except for serotype 5) . The conjugation reaction was stopped by adding the sodium borohydride solution to obtain 0.6 to 1.0 mol of sodium borohydride per mol of saccharide (0 mol of borohydride added for serotype 5). The reaction mixture was incubated for 45 to 60 minutes. Step 3: Ultrafiltration Steps
The reaction mixture was diafiltered in a MWCO 100 kDa ultrafilter with a minimum of 20 volumes of 100 mM potassium phosphate buffer, pH 8.4. The retention of the 100 kDa ultrafilter was diafiltered in a 300 kDa MWCO ultrafilter with a minimum of 20 diamolates of 150 mM sodium chloride at 20 to 24 ° C. The permeate was discarded. Step 4: Sterile Filtration
The 300 kDa MWCO diafiltration retained was filtered through a 0.2 pm filter and filled into borosilicate glass containers in appropriate volumes to test release, in-process controls, and formulation (except serotype 19F). The serotype 19F conjugate was passed through a 0.2 pm filter in a containment tank and incubated at 20 to 24 ° C. After incubation, the conjugate was diafiltered in a 300 kDa MWCO ultrafilter with a minimum of 20 diavolumes of 150 mM sodium chloride at 20 to 24 ° C. The permeate was discarded, and the retentate was filtered through a 0.2 pm filter and filled into borosilicate glass containers in appropriate volumes for the release, in-process controls, and formulation. The final bulk concentrates were stored at 2 to 8o C. EXAMPLE 3: Formulation of a 15-valent Pneumococcal Conjugate Vaccine
The required volumes of bulk concentrates were calculated based on the batch volume and bulk saccharide concentrations. The 15 combined conjugates were further diluted to a target absorption concentration by the addition of a buffer containing sodium chloride and L-histidine, pH 5.8. After mixing sufficiently, the mixture was filtered sterile through a 0.2 µm membrane. The sterile formulated coarse was mixed gently during and after mixing with bulk aluminum phosphate. The formulated vaccine was stored at 2 to 8 ° C.
In an alternative process, the 15 combined conjugates were further diluted to a target concentration by the addition of a buffer containing sodium chloride and L-histidine, pH 5.8. Polysorbate 80 was added to a final concentration of 0.005%, to the diluted buffered conjugate mixture before sterile filtration. Following sterile filtration, the formulated vaccine was stored at 2 to 8o C. Table 1 shows the final composition of the forms with PCV-15 adjuvant and without adjuvant. Table 1: Composition of 15-Valente Pneumococcal Conjugate Vaccine Formulations With Adjuvant and Without Adjuvant
EXAMPLE 4: Immunogenicity Studies
The experiments were designed to assess the immunogenicity of multiple pneumococcal conjugate vaccine formulations in animal models of baby Rhesus monkeys (MRI) and New Zealand White Rabbits (NZWR). The experiments on baby Rhesus monkeys were designed to closely match the recommended program for the pneumococcal conjugate vaccine in the United States, with the baby series given at 2, 4, and 6 months of age. Thus, baby rhesus monkeys were immunized leaving at 2 to 3 months of age and vaccine administered at 2-month intervals. The 4th dose, which is also part of the recommended program for American children, was not administered. Adult rabbits (NZWR) were used to evaluate multiple vaccine formulations. NZWR studies were conducted using two doses of vaccine given in an accelerated immunization regimen (2-week interval). For the preclinical assessment of immune responses, a full human dose was given to the rabbits while the baby monkeys received a half human dose. The rationale for selecting a half human dose for baby monkeys was due to the limitations in the volume that can be given to baby Rhesus monkeys in a single intramuscular site. Evaluation of Serotype-Specific IgG Responses
A multiplexed electrochemiluminescence (ECL) assay was developed for use with rabbit and rhesus monkey serum based on a human assay using Meso Scale Discovery (MSD) technology that uses a SULFO-TAG® label that emits light in electrochemical stimulation. See Marchese et al., 2009, Clin Vaccine Immunol 16: 387-96. Using a dedicated ECL plate reader, an electrical current is placed through the electrodes associated with the plate that result in a series of electrically induced reactions that lead to the luminescent signal. The multi-point configuration used in development and validation was 10 points / reservoir in a 96-reservoir plate format, and each reservoir was coated with 5 ng of pneumococcal polysaccharide (Ps) per point. Two plate formats were used to ensure that cross-reacting polysaccharides (i.e., 6A and 6B, and 19A and 19F) were tested on separate plates. Plate format 1 contained serotypes 3, 4, 6B, 9V, 14, 18C, 19F, and 23F whereas plate format 2 contained serotypes 1, 5, 6A, 7F, 19A, 22F and 33F. Each reservoir also contained two points of bovine serum albumin (BSA) that were used to assess the background reactivity of the assay (ie, the response associated with the serum and secondary antibody labeled in the absence of PnPs). The assay standard (89SF-2), controls, and test sera were diluted to the appropriate levels in phosphate buffered saline (PBS) containing 0.05% Tween 20, 1% BSA, 5 pg / ml C -polysaccharide (CPs), 10 pg / ml of serotype 25 polysaccharide (PnPs25) and 10 pg / ml of serotype 72 polysaccharide (PnPs72) and incubated overnight at 4 ° C (2 to 8 ° C) or at room temperature for 45 minutes. Human antibody reagents and standards were used when testing baby monkey samples whereas anti-rabbit IgG labeled with SULFO-TAG® was used as the secondary antibody when testing rabbit serum samples. Each antigen-coated plate was incubated at room temperature for 1 hour on a shaking platform with blocking agent. The plates were washed with 0.05% PBS-T and 25 pl per reservoir of the pre-absorbed and diluted test sera were added and incubated for 45 min at room temperature on a shaking platform. The plates were washed with 0.05% PBS-T and then goat anti-human IgG secondary antibody labeled with MSD SULFO-TAG® (for rhesus monkey serum) and labeled secondary goat anti-rabbit IgG antibody (for rabbit serum) were added to each reservoir and incubated for 1 hour at room temperature on a shaking platform. The plates were washed with 0.05% PBS-T and 150 pl of Reading Buffer MSD-T 4X (with surfactant) diluted 1: 4 in water added to each reservoir. The plates were read using an MSD Sector Imager Model N2 2400 or 6000. For rabbit studies, the results are presented as geometric mean titers (GMTs) or GMT ratios. For studies with baby rhesus monkeys, the results were expressed as geometric mean concentrations read from a standard curve using the serotype-specific IgG concentrations planned for the human reference standard (89 SF-2). Evaluation of Functional Responses (Opsonophagocytic)
Samples from study 2 of baby rhesus monkeys were tested in a MOPA 4-plexed assay (MOPA-4). See Burton et al., 2006, Clin Vaccine Immunol 13: 1004-9. The assay uses bacterial strains selected for being resistant to one of 4 antibiotics so that the first part of the assay (opsonization and uptake in differentiated HL-60 cells) can be performed with up to 4 serotypes at once. The reading for bacterial death is done in parallel in the presence of each of the 4 antibiotics for which the corresponding strains are resistant in order to determine the titers of death for each specific serotype. The results are expressed as the reciprocal dilution at which 50% death is observed (after interpolation). Statistical Methodology for Pre-Clinical Studies
Both animal models had limitations related to the sample size. Overall, 8 baby monkeys or 8 rabbits were used per study branch. With 8 animals per branch, a critical times difference in the mean geometric titer between the treatment branches of 2.5 times was considered as a significant response threshold. The 2.5-fold difference was determined based on the assumption that for each serotype, the standard deviation of the titers transformed into a natural log within a treatment branch is ln (2). Allowing Cj to indicate the average of the titers transformed into ln in the treatment branch, n the number of animals within the treatment branch, Oj2 the known variation of the titles transformed in ln among the animals within the treatment branch, and adjusting n; = 8 and o; = (Zn (2)) for all i,
Then the value of 2.5 is obtained by solving e ° J 'Dkonde
= Z0_995, and ZO, 995 indicates the inverse of the standard normal cumulative distribution, with a probability of 0.995 (that is, Z0.995 = 2.576). Note that the calculated value of 2.44 is rounded to 2.5 as 2.5 also provides a convenient reciprocal of 0.4. Serotype-Specific IgG Response of Baby Rhesus Monkeys (MRIs) to PCV-15
A pilot immunogenicity study (MRI-1) was conducted to determine whether baby Rhesus monkeys (MRIs) would be a good model in which to evaluate Pn CRM197 polysaccharide conjugate vaccines. The primary goal of the experiment was to determine whether MRIs (like human babies) would be unresponsive to free Pn polysaccharides but would respond well to conjugate vaccines. Groups of 5 MRIs were injected leaving at 2 to 3 months of age with polysaccharide Pn, Prevnar® or PCV-15. Three doses of vaccine were administered intramuscularly (IM) at 2-month intervals, and serotype-specific IgG responses were measured before the first dose and at 1 month after dose 2 and at 1 month after dose 3 using a multi-array electrochemiluminescence (ECL) (data not shown).
The results indicated that the MRIs responded poorly, if not at all, to the free Pn polysaccharide but very well to the conjugate vaccines. The results indicated that the induction of an IgG response to Pn polysaccharides in baby Rhesus monkeys was dependent on the conjugation of the polysaccharides to a carrier protein and therefore was a classic T cell dependent response. Thus, the IRM model was determined to be suitable for evaluating PCV-15 formulations.
A second study (IRM-2) was conducted to evaluate a PCV-15 formulation using a batch conjugation process that minimized free (unconjugated) and unconjugated CRM197. Figure 1 shows the post-dose 2 (PD-2) and post-dose 3 (PD-3) IgG responses for PCV-15 versus Prevnar® for the 7 serotypes contained in Prevnar® (4, 6B, 9V, 14, 18C, 19F, 23F). PD-2 responses to PCV-15 were equivalent to or slightly lower than the corresponding responses to Prevnar® whereas PD-3 responses to PCV-15 were slightly higher than those evoked by Prevnar® for almost all serotypes.
MRI responses to non-Prevnar serotypes in PCV-15 are shown in Figure 2. PD-2 responses to non-Prevnar serotypes in PCV-15 were all at least 10 times higher than baseline IgG concentrations ( pre-vaccination), and titers continued to rise in PD-3.
The results indicate that antibody responses to PCV-15 and Prevnar® were comparable for the 7 common serotypes and that post vaccination responses to PCV-15 were> 10 times higher than the baseline for the 8 added serotypes . Functional Immune Response (Opsonophagocytic) of MRIs to PCV-15
In order to determine whether PCV-15 induced functional antibody responses in baby monkeys, an opsonophagocytic death (OPA) assay was performed on the MRI-2 sera. The responses in pre-vaccination, PD-2, and PD-3 to PCV-15 and Prevnar® are shown in Table 2. The results shown are the GMTs of serum samples from 7 to 8 monkeys per time point tested in duplicate. Percentage responders (that is, those with OPA titles> 8) at time point PD-3 are also shown. PCV-15 induced a high PD-2 GMT for all serotypes except types 1 and 33F. After 3 doses of vaccine, PCV-15 induced high OPA GMTs for each serotype and a 100% OPA response rate for all 15 serotypes contained in the vaccine. Importantly, PCV-15 also induced a good cross-reactive OPA response to serotype 6C, which is not in the vaccine. Prevnar® induced high OPA titers and a 100% response rate for all serotypes contained in this vaccine, but it induced only a weak cross-reactive response to serotypes 6A and 6C in a fraction of monkeys. Table 2: Serotype-specific OPA GMTs in Baby Rhesus Monkeys After Vaccination with PCV-15orPrevnar® (geometric mean titles ng — pre-vaccination, —PD-2. percentage responders PD-3 coiXLum title> 8)
Evaluation of PCV-15 Formulations in New Zgaland White Rabbits
PCV-15 formulations were evaluated in 4 studies in adult New Zealand White Rabbits (NZWRs) using a compressed immunization regimen in which rabbits received a full human dose of vaccine on day 0 and day 14, and serum was collected in days 0, 14 and 28 for the analysis. All studies were compared with Prevnar, and as summarized in Table 3 (Experiments with NZWR 1 to 4).
The results are shown in Table 3 for the post-dose 2 responses of New Zealand white rabbits expressed as a ratio of geometric mean IgG responses for Merck PCV-15 to Prevnar® for serotypes in common between vaccines. Table 3 Post-dose 2 IgG Response Ratios (PCV-15: Prevnar®) of Initial PCV-15 Formulations Tested in NZWR

Serotype-specific IgG responses were generally within 2.5 times of the corresponding responses to Prevnar. An exception was the serotype (23F), which was> 2.5 times lower than that for Prevnar® in 2 of 4 experiments. The times of increase in antibody levels for non-Prevnar® serotypes from Day 0 to Day 28 (after dose 2, PD-2) are summarized in Table 4. Table 4 Times of increase (after dose 2: Pre-dose 1) IgG Responses to Non-Prevnar® Serotypes of Initial PCV-15 Formulations Tested in NZWR
Effect of Dose of Conjugated Polysaccharide Vaccine on Immunogenicity in NZWRs
The immunogenicity of an increased dose (double dose, 2x) of polysaccharide conjugates was also assessed for all serotypes contained in PCV-15 compared to the planned human dose (lx) of the vaccine. For the 2x polysaccharide conjugate formulation, the APA concentration was increased by 1.5x in order to ensure that most of the conjugate would be linked to the aluminum adjuvant. As shown in Table 5, it did not appear to be a significant benefit in increasing the amount of polysaccharide conjugate in the vaccine. The differences in all serotypes were within 2 times, and the geometric mean times ratio (lx PCV-1512X PCV-15 + 1.5X APA) was 1.1. Table 5 Geometric Average IgG Titles in Postdose 2 (95% confidence intervals) with Prevnar®, lx the human dose of PCV-15 or 2x the Human Dose of PCV-15f in NZWR
* Formulated with 1x aluminum adjuvant (APA) t Formulated with 1.5x APA Effect of Aluminum Adjuvant on PCV-15 Immunogenicity in NZWRs
The impact of aluminum adjuvant (APA) on antibody responses was evaluated in a rabbit study. PCV-15 formulated with the planned human dose of APA (PCV-15 lx APA), with the double planned human dose of APA (PCV-15 2x APA), and without any aluminum adjuvant (PCV-15 0x APA), were tested. A group of Prevnar® was also included in the study.
The PD-2 results indicated that doubling the APA concentration had little impact on the serotype-specific IgG response to PCV-15. The times of difference in the title (1x APA / 2x APA) ranged from 0.6 (serotype 6B) to 2.3 (serotype 22F) and the geometric mean 5 times ratio across the 15 serotypes was 1.1. In the absence of aluminum adjuvant, antibody titers appeared lower for many of the serotypes related to PCV-15 with 1x APA. The times of difference in the title (lx / 0x) varied from 0.5 (serotype 5) to 2.9 (serotype 23F) and the ratio in geometric mean times across the 15 serotypes was 1.4. Overall, there did not seem to be a genuine advantage in doubling the level of aluminum adjuvant and there appears to be a disadvantage to eliminating the adjuvant (Table 6) in this animal model.
The PD-2 results indicated that there was a decrease in antibody titers for many of the serotypes in the branch that did not contain Aluminum Phosphate Adjuvant (APA) when compared to PCV-15 containing APA (Figure 3) indicating a requirement for inclusion of an aluminum adjuvant for the ideal immunogenicity of PCV-15 in rabbits. In addition, no benefit was found when doubling the amount of PAC was included in the vaccine (data not shown). Table 6 Post-dose Geometric Average IgG Titles 2 (95% confidence intervals) of PCV-15 Formulated with 1x, 2x or Ox Adjuvant Aluminum (APA) in NZWR
Debate and Conclusions
Preclinical data demonstrate that a PCV-15 formulation (formulated in PAC) is highly immunogenic in two species (baby Rhesus monkeys and rabbits). The specific serotype responses to the 5 PCV-15 were comparable to those evoked by Prevnar® for the 7 serotypes in common between the vaccines. For the 8 new serotypes in PCV-15, there was a robust response evoked in both baby Rhesus monkeys and rabbits, with a> 10-fold increase in IgG responses for all serotypes after 2 doses of vaccine in both species. Experiments with limited dose variation indicated that a 2-fold increase in the amount of polysaccharide conjugates does not result in an increased antibody response. Similarly, a 2-fold increase in the concentration of the aluminum adjuvant did not appear to significantly improve the PCV-15 immunogenicity profile. Elimination of the adjuvant, however, resulted in 15 lower responses for some serotypes suggesting the potential need for an adjuvant in humans. Functional antibody responses (OPA) were evoked by PCV-15 for all 15 serotypes in the vaccine as well as for serotype 6C, which is not a component of PCV-15.

权利要求:
Claims (7)
[0001]
1. Immunogenic composition, characterized by the fact that it comprises: (1) a multivalent polysaccharide-protein mixture having capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F of Streptococcus pneumoniae conjugated to CRM197 and no other polysaccharide-S. pneumoniae protein conjugate of a different serotype; and (2) a pharmaceutically acceptable carrier.
[0002]
2. Immunogenic composition according to claim 1, characterized by the fact that it additionally comprises an adjuvant.
[0003]
3. Immunogenic composition according to claim 2, characterized by the fact that the adjuvant is an adjuvant based on aluminum.
[0004]
4. Immunogenic composition according to claim 3, characterized by the fact that the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
[0005]
5. Immunogenic composition according to claim 4, characterized by the fact that the adjuvant is aluminum phosphate.
[0006]
6. Use of a multivalent polysaccharide-protein conjugate, characterized by the fact that it is for the preparation of an immunogenic composition as defined in claim 1 to induce an immune response to a Streptococcus pneumoniae capsular polysaccharide.
[0007]
7. Use according to claim 6, characterized by the fact that the immunogenic composition is a single dose of 0.5 ml formulated to contain: 2 pg of each saccharide, except for 6B to 4 pg; about 32 pg of CRM197 carrier protein; 0.125 mg of elemental aluminum adjuvant (0.5 mg of aluminum phosphate); 150 mM sodium chloride and 20 mM L-histidine buffer.

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RU2012138368A|2014-03-20|

SG183199A1|2012-09-27|

JP2014012720A|2014-01-23|

CA2788680A1|2011-08-18|

AU2011216095A1|2012-08-09|

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

2019-01-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

2019-06-11| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

2020-08-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

2020-11-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 03/02/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

申请号 | 申请日 | 专利标题

US30272610P| true| 2010-02-09|2010-02-09|

US61/302726|2010-02-09|

PCT/US2011/023526|WO2011100151A1|2010-02-09|2011-02-03|15-valent pneumococcal polysaccharide-protein conjugate vaccine composition|

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