Multivalent meningococcal conjugates and methods for preparing conjugates
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-9956278-B2
Publication Date
2018-05-01
Expiration Date
2033-05-23
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Abstract
Disclosed herein are meningococcal immunogenic conjugates which can elicit immune responses against meningococcal polysaccharides (PS) from groups A, C, W-135, and Y and group B factor H binding protein (fHbp). The disclosed conjugates also exhibit bactericidal activity against meningococcal A, C, W-135, Y, B, and X serogroups. Also disclosed are improved methods for preparing conjugates, such as immunogenic conjugates, including activation of a polysaccharide with a cyanylation agent at about 4° C.
Core Innovation
The invention discloses immunogenic conjugates comprising at least one polysaccharide or protein conjugated to a Neisseria surface protein, such as group B factor H binding protein (fHbp) or Neisserial surface protein A (NspA). These meningococcal immunogenic conjugates can elicit immune responses against polysaccharides from meningococcal groups A, C, W-135, and Y, and can exhibit bactericidal activity against serogroups A, C, W-135, Y, B, and X. The conjugates can be multivalent, including multiple antigenic epitopes or mixtures of distinct polysaccharides conjugated to carrier proteins.
Additionally, the invention provides improved methods for preparing conjugates including immunogenic conjugates, wherein a first moiety such as a polysaccharide or protein is activated with a cyanylation agent at about 2° C. to about 6° C., resulting in a cyanate-activated moiety. This activated moiety is then contacted with a second moiety having amino or hydrazide groups at about 2° C. to 6° C., forming a conjugate comprising at least one C—N bond. These methods improve conjugation yield and facilitate large-scale production by providing longer activation times at lower temperatures (e.g., 4° C.) relative to conventional room temperature, short activation methods.
The problem addressed is that conventional meningococcal vaccines based on capsular polysaccharides have limited efficacy against group B meningococcal serogroups, as their polysaccharides are structurally similar to human tissues and therefore poorly immunogenic. There is also a need for vaccines effective against group X serogroups. Furthermore, previously known chemical conjugation methods required short, non-ideal activation times at room temperature, posing control and yield challenges, especially for large-scale vaccine production. Thus, there remains a need to develop conjugates and improved conjugation methods that elicit protective immune responses against diverse meningococcal serogroups, including group B and X, with better process control and yield.
Claims Coverage
The patent includes six independent inventive features focused on methods of preparing immunogenic meningococcal conjugates involving polysaccharide activation and conjugation to fHbp proteins.
Method for preparing an immunogenic meningococcal conjugate by cyanylation activation at controlled low temperature
The method comprises reacting at least one meningococcal polysaccharide (group A, C, W-135, Y, or combinations thereof) with a cyanylation agent at 2° C. to 6° C. to yield a cyanate-activated polysaccharide, then contacting this polysaccharide with at least one protein comprising a carrier protein (fHbp subfamily A, fHbp subfamily B, or both) at pH 5 to 8 and 2° C. to 6° C., resulting in an immunogenic conjugate containing C—N bonds formed between the polysaccharide and protein.
Method for preparing hydrazide-activated protein prior to conjugation
The method includes reacting the protein with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, or mixtures thereof at 2° C. to 6° C. to produce hydrazide-activated protein before contacting with the cyanate-activated polysaccharide.
Hydrazide activation using carbodiimide-catalyzed reaction
The protein is reacted with hydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazide, or mixtures thereof in the presence of a carbodiimide hydrochloride at pH 6 to 7 to obtain hydrazide-activated protein.
Use of specific carbodiimide for hydrazide activation
The carbodiimide used is 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride.
Selection of cyanylation agents for polysaccharide activation
The cyanylation agents include CDAP (1-cyano-4-dimethylammoniumpyridinium tetrafluoroborate), CPIP (1-cyano-4-pyrrolidiniopyridinium tetrafluoroborate), cyanogen bromide, p-nitrophenyl cyanate (pNPC), or N-cyano-N,N,N-triethylammonium tetrafluoroborate (CTEA).
Use of fHbp fusion proteins as carrier proteins
The protein carrier can be an fHbp fusion protein comprising at least a portion of fHbp1 and at least a portion of fHbp2.
The claims focus on improved methods for preparing meningococcal polysaccharide-protein conjugates using cyanylation activation at low temperatures and conjugation to fHbp carrier proteins, including hydrazide activation of proteins and the use of specific activating agents and fusion proteins, providing enhanced control and yield of immunogenic conjugates.
Stated Advantages
Improved control and convenience of conjugation reactions by activating polysaccharides with cyanylation agents at lower temperatures (about 4° C.) for longer times rather than short activation at room temperature.
Increased yield of polysaccharide-protein conjugates compared to standard room temperature activation methods.
Suitability of improved conjugation methods for large-scale production, facilitating commercial vaccine manufacture.
Avoidance of contamination by cyanide in conjugation reactions by using hydrazide-aldehyde reductive amination without sodium cyanoborohydride.
Documented Applications
Preparation of immunogenic conjugates that elicit immune responses against meningococcal polysaccharides from groups A, C, W-135, Y, and group B factor H binding protein (fHbp), with bactericidal activity against serogroups A, C, W-135, Y, B, and X.
Production of multivalent meningococcal conjugate vaccines comprising different immunogenic-distinct polysaccharides conjugated to fHbp or NspA.
Use of the conjugates and methods to immunize subjects to prevent, inhibit, or treat meningococcal disease caused by various serogroups.
Application of the improved cyanylation conjugation methods for preparing chemical, biochemical, medical, pharmacological, diagnostic and/or therapeutic reagents beyond meningococcal conjugates.
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