Multivalent conjugate vaccines with bivalent or multivalent conjugate polysaccharides that provide improved immunogenicity and avidity
10688170 ยท 2020-06-23
Assignee
Inventors
Cpc classification
A61K2039/64
HUMAN NECESSITIES
A61K2039/6037
HUMAN NECESSITIES
International classification
Abstract
The disclosure describes compositions containing conjugates using novel linkers, bivalent polysaccharide conjugates, and methods of bivalent polysaccharide conjugation in the development of multivalent conjugate vaccines. Conjugation of capsular polysaccharides to carrier proteins is carried out using homo-bifunctional and/or hetero-bifunctional linkers of specific lengths. Incorporation of the linkers and their use in bifunctional linkers induces higher titers of functional antibodies with high avidity, eliciting higher immunologic memory, and reduced carrier protein effect. This provides immunochemically cross-reactive capsular polysaccharides wherein one or more cross-reactive capsular polysaccharides are conjugated sequentially or concurrently to carrier protein using bifunctional linkers bearing the same or different functional groups. Such a linker and the size of the capsular polysaccharides provides an effective multivalent conjugate vaccine with high antibody titers and a reduced carrier effect and results in reduction in the content of the capsular polysaccharide and protein per dose of vaccine which reduces reactogenicity.
Claims
1. An immunogenic composition comprising: first and second group conjugates, wherein: first group conjugates comprise a first collection of monovalent conjugates, wherein each monovalent conjugate comprises a first carrier protein and a capsular polysaccharide of Streptococcus pneumoniae and the first collection includes capsular polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 7F, 8, 10A, 11A, 12F, 14, 17F, 18C, 20, 22F, 23F, 24F, 33F and 35B; and second group conjugates comprise a second collection of bivalent and/or multivalent conjugates, wherein each bivalent or multivalent conjugate comprises a second carrier protein and at least two capsular polysaccharides of Streptococcus pneumoniae, selected from one or more of the groups consisting Streptococcus pneumoniae serotypes 6A, 6B, 6C, and and/or 6D, Streptococcus pneumoniae serotypes 9V, 9N, 9A and 9B, Streptococcus pneumoniae serotypes 15B, 15A, and 15C, and Streptococcus pneumoniae serotypes 19A and 19F, wherein one or more of the capsular polysaccharides are coupled via a bifunctional linker, wherein the multivalent conjugate contains a quantity of carrier protein that is less than a quantity of capsular polysaccharide.
2. The immunogenic composition of claim 1, wherein the second group conjugates are bivalent conjugates.
3. The immunogenic composition of claim 2, wherein the bivalent conjugates comprises two immunologically cross-reactive serotypes represented by the formula PS1-Carrier Protein-PS2, wherein PS1 and PS2 represent different polysaccharides and carrier protein refers to the second carrier protein.
4. The immunogenic composition of claim 2, wherein the bivalent conjugates are conjugated to the same carrier protein sequentially or concurrently.
5. The immunogenic composition of claim 1, wherein the second group conjugates comprise bivalent conjugates and the capsular polysaccharides of the bivalent conjugates have a molecular weight of from 10 kDa to 50 kDa.
6. The immunogenic composition of claim 1, wherein the bifunctional linker further comprises a spacer arm of from 9-40 .
7. The immunogenic composition of claim 1, wherein the first carrier protein and/or the second carrier protein comprises tetanus toxoid, diphtheria toxoid, CRM197, tetanus toxoid fragments (TTHc), N. meningitidis protein PorB, RSV virus proteins, Bordetella pertussis proteins, Pertussis toxoid (PT), adenylate cyclase toxin (ACT), Human Papilloma viral protein antigens, a Human Papilloma viral 69 KDa protein, Human Papilloma virus VLP forms, Hepatitis B virus core antigen, Hepatitis B virus VLP forms, derivatives of HBsAg, and/or combinations thereof.
8. The immunogenic composition of claim 1, wherein a single dose comprises less than 4 micrograms.
9. The immunogenic composition of claim 1, wherein the quantity of the first and second carrier proteins is from 0.5% to 0.8%, by weight, of the conjugates.
10. The immunogenic composition of claim 1, wherein the second group conjugates comprise bivalent conjugates and the capsular polysaccharides of the bivalent conjugates have a molecular weight of from 30 KDa-100 KDa.
11. The immunogenic composition of claim 1, wherein one or more of the monovalent conjugates comprise a linker.
12. The immunogenic composition of claim 1, further comprising at least one adjuvant selected from the group consisting of aluminum salt, calcium phosphate, a liposome of monophosphoryl lipid A (MPLA), saponin QS-21, and a potent TLR7/8 agonist.
13. The immunogenic composition of claim 12, wherein the aluminum salt is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
14. The immunogenic composition of claim 1, wherein the second collection includes Streptococcus pneumoniae serotypes 6A, 6B, 6C, and 6D, Streptococcus pneumoniae serotypes 9V, 9N, 9A and 9B, Streptococcus pneumoniae serotypes 15B, 15A, and 15C, and Streptococcus pneumoniae serotypes 19A and 19F.
15. The immunogenic composition of claim 14, further comprising capsular polysaccharides of one or more serotypes of Haemophilus influenza type a and/or b; Group B Streptococcus, Moraxella catarrhalis lipo-oligosaccharides (LOS), non-typable Haemophilus influenzae (NTHi) and/or Neisseria meningitis.
16. The immunogenic composition of claim 1, wherein the second group conjugates comprise bivalent conjugates and the capsular polysaccharides of the bivalent conjugates have a molecular weight of from 100 KDa-300 KDa.
17. The immunogenic composition of claim 1, wherein administration of a single dose generates a lower immune response to the carrier protein in comparison to administration of a similar conjugate containing only monovalent conjugates of the same polysaccharides.
Description
DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
DESCRIPTION OF THE INVENTION
(12) Streptococcus pneumoniae is a Gram-positive bacterium which can cause diseases such as pneumonia, bacteraemia, meningitis, and acute Otitis media. Pneumococcus is encapsulated with a chemically linked polysaccharide which results in serotype specificity. At least 90 pneumococcal serotypes are known of which about 23 account for 90% of invasive diseases. The protection against invasive pneumococci disease is related to the antibody specific to the capsular polysaccharide, the protection is therefore serotype specific.
(13) It was surprisingly discovered that multivalent S. Pneumoniae conjugate vaccine comprising of a linker between the carrier protein and the polysaccharide to form two groups of conjugates, wherein group one comprises monovalent bacterial capsular polysaccharide conjugates and the other group comprises multivalent carrier protein conjugates provides substantially improved results. Specifically, the bivalent conjugates and bivalent unimolecular conjugates are preferably synthesized by the reaction between carrier protein and bifunctional linkers attached to cross reactive S. Pneumoniae serotypes. Results achieved are substantially improved compared to vaccines containing multivalent S. Pneumoniae conjugate vaccine containing monovalent bacterial capsular polysaccharide conjugates with the same number of serotypes with a direct conjugation between the two instead of a linker.
(14) One embodiment of the invention is directed to multivalent conjugate vaccines comprised of bivalent-polysaccharide protein conjugates with enhanced immunogenicity. Bivalent conjugates with general structure PS1-carrier protein-PS2 have higher immunogenicity compared to similar monovalent conjugates wherein PS1 and PS2 are two different serotype polysaccharides from gram-negative and gram-positive bacterial pathogens. By developing a bi-valent conjugate vaccine, the efficacy of the vaccine increases and carrier immunogenicity is reduced. The chemistry disclosed herein substantially increases the conjugates immunogenicity, at the same time reduces carrier protein load.
(15) Another embodiment of the invention is directed to vaccines with lower molecular weight polysaccharides and longer arm bifunctional linkers preferably with enhanced immunogenicity. Another embodiment of the invention is directed to providing higher immunogenicity and avidity of bivalent conjugates as well as lower carrier protein immunogenicity. Another embodiment of the invention is directed to reducing conjugate vaccine dose with higher immunogenicity.
(16) As disclosed herein, four parameters have been introduced to minimize the disadvantages of conventional vaccines: Polysaccharide size is preferably 10-50 KDa. Cross-reactive polysaccharides concurrent conjugation to carrier protein. Two or more cross reactive serotypes are conjugated concurrently with carrier proteins. A long hetero- or homo-bifunctional spacer arm preferably of from 2-40 (also 2-40 , 4-40 , 10-40 , 20-40 , 9-20 , 5-20 , 5-30 ).
(17) These four parameters taken together are profoundly effective to increase the conjugates polysaccharide/protein ratio, to reduce carrier protein load, and to provide several folds of increase in immunogenicity and avidity.
(18) The present invention is directed to polysaccharide-protein conjugates with enhanced immunogenicity displaying significantly high antibody titers. The carrier protein is obtained from, for example, tetanus toxoid, diphtheria toxoid, CRM197, tetanus toxoid fragments (TTHc), N. meningitidis protein PorB, RSV virus proteins, B. Pertussis proteins like pertussis toxoid (PT), adenylate cyclase toxin (ACT), 69 KDa protein and Human Papilloma viral protein antigens or its VLP form, Hepatitis B core antigen or its VLP form or derivatives of HBsAg, and other conventional carriers. Polysaccharide fragment is obtained from group of group of gram positive bacteria and gram-negative bacteria, preferably from immunochemically cross-reactive polysaccharides of S. Pneumoniae. The present invention is also directed to a process of preparing the polysaccharide-protein conjugates in which carrier protein reacts with cleaved and depolymerized polysaccharide fragments of optimum chain length.
(19) Immunogenic compositions of the present invention provide for appropriate level and improved protection against S. pneumoniae serotypes not found in PREVNAR-13 , and SYNFLORIX-10.
(20) Bivalent conjugates with cross-reactive polysaccharides of S. Pneumoniae serotypes (6A/6B, 9V/9N, 15A/15B and 19A/19F and similar cross-reactive serotypes) with short chain molecular size (10-50 KDa) was used to prepare 16-26-valent pneumococcal CPS conjugate vaccine in the present study. Pneumococcus type 6A and 6B polysaccharide was used as the model cross-reactive CPSs. CRM197 was used as the carrier protein for its clinical acceptance.
(21) Multivalent monoconjugates have also been prepared using shorter PS chain length (0-50 KDa), long spacer arm (9-40 ) with homo or hetero-bifunctional PEG or non-PEG linker with carrier protein CRM197.
(22) CPS was activated either by oxidation or by cyanylation chemistry and oxidized by sodium periodate and introduced with either-reactive aldehyde or isothiocyanate (OCN) groups in CPS.
(23) Two strategies (short and long linker, short and long CPSs) were used to introduce, respectively. Physicochemical and immunological characteristics of the bivalent conjugates vaccines were then investigated independently or combining with multivalent conjugate formulation.
(24) The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.
EXAMPLES
Example 1 Polysaccharide Size Reduction, Activation and Conjugation Process for Multiple S. pneumoniae Serotypes-1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 35B
(25) 6A and 6B Polysaccharide
(26) 100 mg each of capsular polysaccharides of S. pneumoniae 6A and 6B is dissolved in 10 ml of aqueous solution containing 10 mM of Acetic acid or 0.1 M HCl at pH 2.5-3.0 and hydrolysis is carried out by maintaining the solution at a temperature of 60-85 C. for a period of 60-120 mins. The so-obtained oligosaccharides after neutralization, diafiltered using 3-10 KDa TFF Centricon filters. Upon .sup.1H NMR analysis (
(27) CPS (50 mg) moiety (native polysaccharides of size between 200-500 KDa or size-reduced polysaccharides of size between 10-50 KDa) were activated cyanylation reagents commonly used in activation process (Table 1). Polysaccharide molecular size distributions were determined using SEC-HPLC (Shodex SB-405 and SB-406 SEC columns) with analysis using (10-1000 KDa) Pollulan mixture as reference standard (Pollulan standards from Shodex, USA).
(28) Short spacer arm was introduced to PS by reaction with 5-8-fold molar excess of ADH (Sigma) at pH 5.6-6.0 for 3-5 hr. Long spacer arm (bifunctional linker or long 4-arm linker) was introduced into PS by reaction with 5-10-fold molar excess of at pH 5.6-6.0 for 3-5 h.
(29) TABLE-US-00001 TABLE 1 Polysaccharide size distribution (KDa) used for conjugation PS Polysaccharide KDa 6A 10-30 KDa 6B 20-50 KDa 15B 20-40 KDa 18C 20-50 KDa 22F 10-30 KDa
(30) Activated PS is further derivatized with short arm linker (adipic acid di-hydrazide, ADH, 174.2 g/mole), one more spacer arm linkers with varying size from 2-4 to 8-20 (600 g/mol-3.5 g/mole).
(31) Homo or hetero-bifunctional PEG linkers with diamine functional groups attached, e.g. NH2-PEG0.6KNH2, NH2-PEG3.5KCOOH (Table 2).
(32) TABLE-US-00002 TABLE 2 Short and long chain linker used for polysaccharide or carrier protein derivatization used (several other linkers either in pegylated form or non-pegylated form have also been used) Linker No. Linker Structure Chemical Structures/KDa or used 1 NH.sub.2PEGNH.sub.2/NHS H.sub.2N(CH.sub.2CH.sub.2O)nCH.sub.2CH.sub.2NH.sub.2 1K and 3.5K 2 NHS/NH.sub.2PEGCOOH
(33) Two aliquots of 2 ml each of the derivatized CPS (10 mg/ml) were mixed with 1 ml aliquot of the two CRM197 protein samples (10 mg/ml) at 4 C. for 8-12 hrs. The conjugates with long and short spacer arm were purified by a 100-300 KDa Centricon filters (EMD Millipore) (Table 3).
(34) TABLE-US-00003 TABLE 3 Physicochemical Characterization of mono-valent Conjugates Activated PS KDa Conjugate KDa PS:Protein Free PS by SEC-HPLC by SEC-HPLC ratio PS % 6A 10-30 KDa, 200-300 KDa >200-300, >2500 0.5-2, 1-2 <2 6B 20-50 KDa, 200-400 KDa >300-500, >2500 0.5-2, 1-2 <1 15B 20-40 KDa >300-500 0.5-2, 1:1 <1 18C 20-50 KDa >300-500 0.5-2, 1:1 <2 22F 10-30 KDa >200-300 0.5-2, 1:1 <1 Note: Internal std. for KDa determination of PS for SEC-HPLC:Pullulan std. mixture (2 KDa-2500 KDa).
Example 2 Activation of Size Reduced Polysaccharides of Different Molecular Weights Oligosaccharides of Different Molecular Weights Synthesized as Described in Example 1 were Activated. Cyanylation Reagents Commonly Used in Activation Process
(35) CDAP (1-Cyano-4-dimethylaminopyridinium tetrafluoroborate (Sigma Aldrich, USA)) cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) or cyanogen bromide (CNBr) and coupling carrier protein (see
(36) Polysaccharide solution (10 mg/ml) was incubated with 10 mg/ml CDAP (100 mg/ml in acetonitrile) in 2M NaCl or 200-300 mM bicarbonate buffer at RT for 4-6 minutes. pH was maintained at 10-10.5 using either 1N NaOH or 1N HCl. Then, pH was adjusted to 8.1-8.2, pegylated linkers (Hz-PEG-HZ) were allowed to react with CDAP treated PS. For 8-12 hrs at RT. The reaction mixtures were depth filtered followed by 100-300 KDa cutoff centricon fiters 5-8 times using 150 mM NaCl.
(37) Derivatization of Activated Size Reduced Polysaccharides
(38) Activated oligosaccharides were further derivatized with short chain homo-bifunctional hydrazide linker. Typical reagent was adipic Acid di-hydrazide, ADH, Molecular weight 174.2 g/mole). Homo or hetero-bifunctional PEG linkers bearing di-amine, di-hydrazide, or amine or hydrazide-carboxylic acid/aldehyde functional groups, e.g. NH2-PEG(1K-3.5K)NH2, HZ-PEG(1-3.5K)HZ, NH2-PEG3.5KCOOH were used. (Table 2). Several other homo- or hetero-bifunctional spacer arms can also be used for derivatization (Table 2). Short spacer arm was introduced to oligosaccharide by reaction with 5-8 fold molar excess of adipic acid di-hydrazide (Sigma) at pH 5.8-6.0 for 3-5 hr. long chain linker (bifunctional linker or long tetra functional linker (Table 2), No. 12 four arm linker) was introduced into Polysaccharide by reaction with 5-10-fold molar excess of the linker to the oligosaccharide at pH 5.8-6.0 for 3-5 hrs. at RT.
(39) Derivatization of Carrier Protein with Short or Long-Linkers
(40) Carrier protein CRM197 was further derivatized with short chain homo-bifunctional hydrazide linker. Typical reagent was adipic Acid di-hydrazide, ADH, molecular weight 174.2 g/mole). Homo or hetero-bifunctional PEG linkers bearing di-amine, di-hydrazide, or amine or hydrazide-carboxylic acid/aldehyde functional groups, e.g., NH2-PEG(1K-3.5K)NH2, HZ-PEG(1-3.5K)HZ, NH2-PEG3.5KCOOH were used. (Table 2). Several other homo- or hetero-bifunctional spacer arms can also be used for derivatization as listed in Table 2). Short spacer arm was introduced to carrier protein CRM197 by reaction with 5-8 fold molar excess of adipic Acid di-hydrazide (Sigma) at pH 5.8-6.2 in 300-600 mM MES buffer for 3-5 hr at RT. Long chain linker (bifunctional linker or long tetra functional linker (Table 2, No. 12 four arm linker) was introduced into carrier protein by reaction with 5-10-fold molar excess of the linker to the oligosaccharide at pH 5.8-6.2 in 300-600 mM MES buffer for 3-5 hr at RT (room temperature).
Example 3 Cross-Reactive Polysaccharide Serotypes Activation and Attachment of Short or Long-Spacer Arm Linkers (Serotypes of Interest are 6A/6B, 9V/9N, 15A/15B and 19A/19F or any Other Cross-Reactive Serotypes)
(41) Activation of the oligosaccharide derived from the capsular polysaccharide of S. Pneumoniae Type 6A and 6B conjugation with CRM197 and introduction of the primary amino groups to the oligosaccharides concurrently.
(42) Native or size reduced polysaccharide of serotype 6A and 6B (200-400 KDa) were conjugated using the same procedure as described in Examples 1 and 2.
(43) The oligosaccharides mixture thus obtained as reported in Example 1 are dissolved in WFI, to an end concentration of 10 mg/ml. At the end of the reaction, the Oligosaccharide are purified by diafiltration using 3-10 KDa Centricon filters.
(44) The Oligosaccharides into which the amino groups have been introduced are diluted to a concentration of 10 mg/ml in an aqueous solution of DMSO (at 20-30% v/v) to DMSO containing ADH short linker or long spacer arm linkers in molar excess relatively to the amino groups introduced into the oligosaccharide (usually 5-10:1). The reaction was carried out by keeping the solutions at RT for a time of 4-12 hours. At the end of the period, oligosaccharide was again purified using 3-10 KDa Centricon filters.
Example 4 Synthesis of Pneumococcal Polysaccharide Monovalent Conjugates
(45) Two separate aliquots of same or differently size reduced and derivatized size reduced Polysaccharides (with short spacer arm ADH and Long spacer arm HZ-PEG-HZ) as synthesized in example 3 (10 mg/ml) were mixed with 1 ml aliquot of the CRM197 protein sample (10 mg/ml) at 4 C. for 8-12 hrs. The conjugates containing both long and short chain linkers were purified using 100-300 KDa centricon filters (EMD Millipore). Each monovalent conjugates were assayed for total polysaccharide content by either anthrone or uronic acid assay, total protein content by BCA or Lowry assay (Table 4).
(46) All other cross-reactive Polysaccharide conjugates are made using the same procedure as above.
(47) TABLE-US-00004 TABLE 4 Physicochemical Characterization of Bi-valent Conjugate of general structure 6A-CRM197-6B Oligosac- Free Activated charide:Protein Oligosac- Oligosac- ratio charide % PS charide KDa Conjugate KDa (Weight ratio) by weight 6A 100-300 KDa >200-300 KDa, 0.5-2, 1-2 <2 >2500 KDa 6B 200-400 KDa >300-500 KDa, 0.5-2, 1-2 <1 >2500 KDa 6C 200-400 KDa >300-500 KDa, 0.5-2, 1-2 <1 >2500 KDa 15B 100-300 KDa >300-500 KDa, 0.5-2, 1:1 <1 >1500 KDa 15A 100-300 KDa >300-500 KDa, 0.5-2, 1:1 <1 >1500 KDa 18C 100-300 KDa >300-500 KDa, 0.5-2, 1:1 <2 >1500 KDa 22F 100-300 KDa >200-300 KDa, 0.5-2, 1:1 <1 >1000 KDa 1 100-300 KDa >200-300 KDa, 0.5-2, 1-2 <2 >2500 KDa 3 200-400 KDa >300-500 KDa, 0.5-2, 1-2 <1 >2500 KDa 4 100-300 KDa >300-500 KDa, 0.5-2, 1:1 <1 >1500 KDa 7F 100-300 KDa >300-500 KDa, 0.5-2, 1:1 <2 >1500 KDa 9V 100-300 KDa >200-300 KDa, 0.5-2, 1:1 <1 >1000 KDa 9N 100-300 KDa >200-300 KDa, 0.5-2, 1:1 <1 >1000 KDa 14 100-300 KDa >200-300 KDa, 0.5-2, 1-2 <2 >2500 KDa 18C 200-400 KDa >800 KDa, 0.5-2, 1-2 <1 >2500 KDa 19A 100-300 KDa >300-500 KDa, 0.5-2, 1:1 <1 >1500 KDa 19F 100-300 KDa >300-500 KDa, 0.5-2, 1:1 <2 >1500 KDa 23F 100-300 KDa >200-300 KDa, 0.5-2, 1:1 <1 >1000 KDa 33F 100-300 KDa >200-300 KDa, 0.5-2, 1-2 <2 >2500 KDa Note: Internal std. for SEC-HPLC (KDa):Pollulan std mixture (2 KDa-1200 KDa)
Example 4 Investigational Formulation of 16V- or Higher Valent Pneumococcal Conjugate Vaccine
(48) Pneumo Polysaccharide-CRM197 conjugates for serotypes containing 1, 3, 5, 7F, 14, 15B, 18C, 22F, 23F, 33F, 35B and cross-reactive polysaccharide conjugates 6A, 6B, 9V, 9N, 15A, 15B, 19A, and 19F were combined to yield final antigen concentration of 4.0 g PS/mL. Sodium chloride (150 mM) solution, 10-20 mM Histidine, Succinic acid and 0.001% Tween-20 was also used during the formulation process as diluent, and aluminum phosphate (Adju-Phos, Brenntag, USA) was used as investigational adjuvant. 16-V conjugate was aseptically filled in 2 mL sterile vials. PNEUMOVAX (Merck, USA) or PREVNAR-13 (Pfizer, USA) was used as two control commercial vaccine formulation.
Example 5 Immunogenicity Studies of Conjugates
(49) A New Zealand white rabbit model (NZW) was selected in this work to compare the immunogenicity of the Pneumo PS-CRM197 conjugates. Rabbits from all groups (16-V {valent}, PREVNAR-13, and PNEUMOVAX) were examined for clinical signs before and after immunization periods. For all groups, pre-immunization, booster dose (7 and 14-days) and terminal bleed (28 days) were collected and aliquoted and store at minus 80 C. until use. Multiplexed Immunogenicity assay for the determination of Total IgG were performed according to the standard protocol using reference standard serum 007 (CBER, FDA, USA). Reference serum and rabbit serum were diluted and pre-adsorbed for cross-reacting antibodies by treatment with pneumococcal CWPS and either 22F PS or 25 PS. Human monoclonal anti-polysaccharide antibodies (Pamlico Biopharma, USA) were used for total IgG estimation. Bio-Plex 200 (Bio-Rad). Multiplex reader was used as per manufacturer's instructions (see
Example 5 S. Pneumoniae Cross-Reactive Capsular Polysaccharide Serotypes Activation and Attachment of Short and Long-Spacer Linkers
(50) Serotypes of 6A/6B, 9V/9N, 15A/15B and 19A/19F which are cross-reactive serotypes are used for the synthesis of bi-valent conjugates containing capsular poly saccharides and carrier protein. Bivalent conjugates by definition contain two capsular polysaccharide attached to CRM 197 simultaneously or concurrently.
(51) Activation of the size reduced polysaccharide derived from the capsular polysaccharide of S. Pneumoniae Type 6A and 6B, conjugation with CRM197 and introduction of the primary amino or hydrazide groups to the oligosaccharides carried out concurrently.
(52) Native polysaccharides or size reduced oligosaccharide of serotype 6A and 6B (200-500 KDa) were conjugated using the same procedure as described in Example 1-4.
(53) The size reduced polysaccharides mixtures thus obtained were dissolved in water for injection, so that the final concentration was 10 mg/ml. The size reduced polysaccharides into which the amino or hydrazide groups were introduced were diluted to a concentration of 10 mg/ml in an aqueous solution of dimethyl sulfoxide (DMSO) so the percentage of DMSO was in the range of 20-30% (v/v). This was added to DMSO containing short chain linker such as ADH or long chain linkers as described in Table 2 in molar excess relatively to the amino/hydrazide groups introduced into the size reduced polysaccharides (usually 5:1 or 10:1), more specifically 8:1.
(54) The reaction was carried out at room temperature for a duration of 4-12 hours. At the end of the reaction period, the reaction product was again purified using 3-10 KDa Centricon filters.
Example 6 Simultaneous or Concurrent Conjugation of S. pneumoniae Oligosaccharides of Type 6A and Type 6B with CRM197 Carrier Protein as Bivalent Conjugates Manufacturing
(55) The aqueous solution containing 15 mg/ml of CRM197, was added to DMSO containing the linker attached oligosaccharide (20-30% in water) derived from the capsular polysaccharide of S. pneumoniae Type 6A. The ratio of linker attached oligosaccharide to CRM197 was selected from 1:1, 2:1, 1:2. The mixture so obtained was kept, under mild stirring, at room temperature for 8-12 hrs. At the end of said time, the solution containing the derivatized oligosaccharide derived from the capsular polysaccharide of S. Pneumoniae 6B was added. The molar ratio of capsular polysaccharide of S. Pneumoniae 6B to the CRM197, was selected from 1:1, 2:1, 1:2). The resulting mixture was kept for 8-12 hrs at room temperature (Table 5). The conjugation reaction can also be carried out by adding, at the same time (concurrently), to the CRM197-containing solution, the two-activated oligosaccharide respectively derived from the capsular polysaccharide of S. pneumoniae Type 6A and from the capsular polysaccharide of S. pneumoniae Type 6B. The oligosaccharide-protein conjugates so obtained were dialyzed using 100-300 KDa dialysis membrane (Spectrum lab, USA), conditioned in 0.01 M phosphate buffer containing 0.2M NaCl (pH=6.6-7.0) and finally filtered through a 0.22 m filter.
(56) All other cross-reactive Polysaccharide conjugates were made using the same procedure as used above. Reaction sequences are depicted in
(57) TABLE-US-00005 TABLE 5 Comparisons of PS Contents Activated Total Poly- Free Bivalent oligosac- saccharide oligosac- Oligosac- charide Protein ratio charide % charide KDa Conjugate KDa by weight by weight 6A-6B 100-300 2.0:1.5 2-1.5 (1:0.75) <2 6A-6B 100-300, >1200-2500 KDa 2-1.4 (1:0.7) <3 300 19A-19F.sup. 100-300 >500-800 KDa 2-1.6 (1:0.8) <2 15A-15B 100-300, >500-1000 KDa 2-1.3 (1:0.65) <3 300 9V-9N 100-300, >500-1000 KDa 2-1.3 (1:0.65) <3 300
Example 7 Investigational Formulation of 18-Valent or Higher Valent Pneumococcal Conjugate Vaccine
(58) Pneumococcal Polysaccharide-CRM197 conjugates for serotypes containing 1, 3, 5, 7F, 14, 18C, 22F, 23F, 33F, 35B (10 serotypes Polysaccharides) and cross-reactive polysaccharide conjugates of (6A, 6B), (9V, 9N), (15A, 15B) and (19A, 19F) (8 serotypes) were combined to yield final polysaccharide concentration of 2.2-4.4 g PS/mL (1.1-2.2 g/human dose, 0.5 mL). Sodium chloride (150 mM) solution, 10-20 mM histidine, 20 mM HEPES or MOPS buffer and 0.001% Tween-20 was also used during the formulation process as diluent, and aluminum phosphate (Adju-Phos, Brenntag, USA) was used as investigational adjuvant.
(59) 18-valent or higher valent (>20V-24V) conjugate was aseptically filled in 2 mL sterile vials. PNEUMOVAX (Merck, USA) and/or PREVNAR-13 (Pfizer, USA) were used as controls.
Example 9 Immunogenicity Studies of the Conjugates
(60) A New Zealand white rabbit model (NZW) was selected in this work to compare the immunogenicity of the Pneumococcal PS-CRM197 conjugates. Rabbits from all groups (18 or higher-Valent conjugates, PREVNAR-13, Pfizer and PNEUMOVAX-23 (Merck USA) were examined for serological titers before and after immunization periods. For all groups, pre-immunization, booster dose (7 and 14-days) and terminal bleed (28 days) were collected and aliquoted and store at 80 C. until use. Immunogenicity assay for the determination of Total IgG were performed according to the standard protocol using reference standard serum 007 (CBER, FDA, USA). Reference serum and Rabbit serum were diluted and pre-adsorbed for cross-reacting antibodies by treatment with Pneumococcal CWPS and non-vaccine serotype 25 PS. Human/rabbit/mouse monoclonal anti-polysaccharide antibodies were used for total IgG estimation. Bio-Plex 200 (Bio-Rad) reader were used as per the manufacturer's instructions.
(61) Immunogenicity of the conjugates, i.e. capsular polysaccharide specific antibodies (total IgG) were measured using bead-based ELISA assay method were given in Table 6. Total IgG values were compared head to head with PREVNAR-13 in rabbit immunogenicity data. 14-day data shows significant increase in titer in IVT-18V-1 vaccine compared to PREVNAR-13 vaccine. Similarly, IVT-18V-1 data has significant booster on IgG values as compared to PREVNAR-13 (Table 6).
(62) TABLE-US-00006 TABLE 6 Capsular Polysaccharides specific antibodies (Total IgG in g/ml) using Multiplex bead-based ELISA assay for 18V-monovalent conjugate vaccines PREVNAR- (IgG) 14 (IgG) 28 IVT-18V- (IgG) 14 (IgG) 28 13 2.2 day/zero day/Zero 1 2.2 day/Zero day/Zero g/dose day day g/dose day day 1 45 350 1 375 1500 3 47 200 3 48 480 6A 188 560 6A 775 3775 6B 165 780 6B 662 3662 18C 50 280 18C 306 3560 19A 45 235 19A 233 2500 19F.sup. 29 290 19F.sup. 72 720 4 49 230 4 150 750 5 186 700 5 550 3550 .sup.7F 180 680 .sup.7F 332 3860 9V 52 520 9V 212 2400 9N 9N 200 2200 14.sup. 85 400 14.sup. 272 2890 15A 15A 672 3900 15B 15B 750 4000 18C 175 800 18C 550 5500 22F.sup. 22F.sup. 1000 8000 23F.sup. 53 450 23F.sup. 212 2420 Note: IVT-18V == 18-V conjugate vaccine (monovalent conjugates mixed together); 9N, 15A, 15B, 22F and 23F serotype are not present in PREVNAR-13, so IgG values not measured; 18-V formulation as monovalent conjugates were prepared using 2.2 g for each serotype except 4.4 g of 6B conjugate. Sodium chloride (150 mM) solution, 10-20 mM histidine, 20 mM HEPES or MOPS buffer and 0.001% Tween-20 was also used during the formulation process as diluent, and aluminum phosphate (Adju-Phos, Brenntag, USA) was used as investigational adjuvant; capsular polysaccharides antibodies (total IgG) using bead-based ELISA: 18-V conjugate vaccine formulation-2 (IVT-18V-2): 10-V formulation as monovalent conjugates and remaining 8-V added as bivalent-conjugates which includes 6A/6B, 9V/9N, 15A/15B and 19A/19F. (vaccine dose used as 2.2 g for each serotype except 4.4 g of 6B) 10-V formulation as monovalent conjugates and remaining 8-V added as bivalent-conjugates which includes 6A/6B, 9V/9N, 15A/15B and 19A/19F. 6A-6B bivalent unimolecular conjugates are used as 2.2 g/dose, remaining bivalent conjugates are used as 2.2 g/dose. Sodium chloride (150 mM) solution, 10-20 mM histidine, 20 mM HEPES or MOPS buffer and 0.001% Tween-20 was also used during the formulation process as diluent, and aluminum phosphate (Adju-Phos, Brenntag, USA) was used as investigational adjuvant.
(63) Immunogenicity of the conjugates, capsular polysaccharide specific antibodies (total IgG) were measured using bead-based ELISA assay method were given in Table 7. Total IgG values were compared head to head with PREVNAR-13 in rabbit immunogenicity data. 14-day data shows significant increase in titer in IVT-18V-2 vaccine compared to PREVNAR-13 vaccine. Interestingly, IVT-18V-2 total IgG data for bivalent conjugates serotypes (for example. 6A/6B, 9V/9N, 15A/15B, and 19A/19F) has significant booster on IgG values as compared to IVT-18V-1 formulation with monovalent conjugates. Therefore, it can be concluded that Bivalent conjugates has better immunogenicity in comparison to monovalent conjugates (Table 7). Therefore, IVT-18V-2 conjugate vaccine formulation has superior immunogenicity not only against PREVNAR-13 but also against IVT-18V-1 formulation. Polysaccharide conjugated with either 1-3.5K linker (HZ-PEG-HZ) elicits much higher immunogenicity in compared to short linker (ADH) or no linker conjugates as in PREVNAR-13.
(64) TABLE-US-00007 TABLE 7 Capsular Polysaccharides antibodies (total IgG) using Multiplex bead-based ELISA PREVNAR- IVT-18V- 13 2.2 Ratio 14 Ratio 28 2 2.2 Ratio 14 Ratio 28 g/dose day/0 day day/0 day g/dose day/0 day day/0 day 1 45 350 1 375 1500 3 47 200 3 50 530 6A 188 560 6A/6B 875/762 4375/4662 6B 165 780 18C 50 280 18C 316 3600 19A 45 235 19A/19F.sup. 300/198 3500/2700 19F.sup. 29 290 4 49 230 4 180 1000 5 186 700 5 550 3600 .sup.7F 180 680 7F 360 4100 9V 52 520 9V/9N 350/300 3400/3200 9N 14.sup. 85 400 14 310 32000 15A 15A/15B 872/850 5900/5600 15B 18C 175 800 18C 600 6800 22F.sup. 22F 1020 8150 23F.sup. 53 450 23F 300 3200 Note: 1VI-18V-2 = 10-monovalent conjugates and 4 bivalent conjugates mixed together; 18-V conjugate vaccine formulation (IVT-18V-3): 10-V formulation as monovalent conjugates used as 2.2 g/dose and remaining 8-V added as bivalent-conjugates which includes 6A/6B, 9V/9N, 15A/15B and 19A/19F used as 1.1 g/dose, except 6B 2.2 g/dose.
(65) Immunogenicity of the conjugates, i.e. capsular polysaccharide specific antibodies (total IgG) were measured using Multiplex bead-based ELISA assay method were given in Table 8. Total IgG values were compared head to head with PREVNAR-13 in rabbit immunogenicity data. 14-day data shows significant increase in titer in IVT-18V-3 vaccine compared to PREVNAR-13 vaccine. Interestingly, IVT-18V-3 formulations with lower dose (2.2 vs 1.1 ug dose), total IgG data for bivalent conjugates serotypes (for example. 6A/6B, 9V/9N, 15A/15B, and 19A/19F) has comparable IgG values as compared to IVT-18V-2 formulations for bivalent conjugate serotypes. Therefore, it can be concluded that bivalent conjugates has better immunogenicity in comparison to monovalent conjugates with lower dose. Therefore, IVT-18V-2 conjugate vaccine formulation has superior immunogenicity not only against PREVNAR-13 but also against IVT-18V-1 formulation. Polysaccharide conjugated with either 1-3.5K linker (HZ-PEG-HZ) elicits much higher immunogenicity in compared to short linker (ADH) or no linker conjugates as in PREVNAR-13 (Table 8).
(66) TABLE-US-00008 TABLE 8 Total IgG data for bivalent conjugates serotypes PREVNAR- IVT-18V- 13 2.2 Ratio 14 Ratio 28 2 2.2 Ratio 14 Ratio 28 g/dose day/0 day day/0 day g/dose day/0 day/0 day 1 45 350 1 375 1500 3 47 200 3 50 530 6A 188 560 6A/6B 825/860 4275/4900 6B 165 780 18C 50 280 18C 316 3600 19A 45 235 19A/19F.sup. 275/250 3400/3000 19F.sup. 29 290 4 49 230 4 180 1000 5 186 700 5 550 3600 .sup.7F 180 680 7F 360 4100 9V 52 520 9V/9N 320/380 3300/3800 9N 14.sup. 85 400 14 310 32000 15A 15A/15B 790/900 5800/6200 15B 18C 175 800 18C 600 6800 22F.sup. 22F 1020 8150 23F.sup. 53 450 23F 300 3200 Note: 1VI-18V-3 = 10-monovalent conjugates and 4 bivalent conjugates mixed together.
(67) Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims. Furthermore, the term comprising of includes the terms consisting of and consisting essentially of.