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Meningococcal polysaccharide conjugate vaccine treating heterobifunctional reagent as conjugation bridge, and its preparation method

A combination vaccine and meningitis technology, applied in the field of biomedicine, can solve the problems of reducing polysaccharide-protein binding efficiency, polysaccharide and carrier protein self-crosslinking, limiting the development of polysaccharide-conjugated vaccines, etc., to improve immunogenicity and reduce space Shielding effect, benefit to quality control effect

Active Publication Date: 2013-05-08
华兰生物疫苗股份有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the above-mentioned traditional polysaccharide-protein binding technology has the following disadvantages: (1) The polysaccharide-AH derivative will continue to react with the polysaccharide activated by cyanogen bromide to form a self-polymer of the polysaccharide, which reduces the polysaccharide-protein binding efficiency; (2) EDAC mediates the combination of ADH-derived polysaccharides and carrier proteins, and at the same time, it is easy to cause self-crosslinking of polysaccharides and carrier proteins, thereby reducing the binding efficiency of polysaccharides and proteins; (3) Both polysaccharides and carrier proteins are large organisms The molecule is connected by ADH with only 6 carbon atoms in the middle. The structure of polysaccharides and proteins is bound to influence each other, making some important epitopes of polysaccharides easily shielded by proteins, thereby reducing the immunogenicity of polysaccharides
These deficiencies limit the further development of polysaccharide conjugate vaccines

Method used

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  • Meningococcal polysaccharide conjugate vaccine treating heterobifunctional reagent as conjugation bridge, and its preparation method
  • Meningococcal polysaccharide conjugate vaccine treating heterobifunctional reagent as conjugation bridge, and its preparation method
  • Meningococcal polysaccharide conjugate vaccine treating heterobifunctional reagent as conjugation bridge, and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of polysaccharide conjugate vaccine

[0036] Preparation of polysaccharide conjugate vaccine without PEG (PS-TT), polysaccharide conjugate vaccine with PEG2K as bridge (PS-P2K-TT), and polysaccharide conjugate vaccine with PEG5K as bridge (PS-P5K-TT) react as figure 1 shown. Dissolve 10 mg of group Y meningococcal capsular polysaccharide in 2 ml of normal saline, add 20 μL of cyanogen bromide solution (w / v 50%) for activation, and react at room temperature for 1 hour. During activation, the pH of the solution was maintained at 10.5 with 0.5M NaOH. After the activation, the pH value of the solution was adjusted to 8.5 with 0.5M hydrochloric acid to terminate the reaction. Subsequently, 10 mg N-2-aminoethyl-maleimide, 60 mg amine-PEG2K-maleimide (PEG molecular weight 2 kDa) and 75 mg amine-PEG5K-maleimide (PEG molecular weight 5 kDa) were added separately ). React overnight at room temperature. After the reaction, use a filter membrane with a ...

Embodiment 2

[0038] Example 2: Characterization of three polysaccharide conjugate vaccines

[0039] The polysaccharide-protein combination product involved in Example 1 was separated and purified with a Superdex200 gel filtration column (2.6cm×60cm), the eluent was 20mM phosphate buffer (pH7.4), and the flow rate was 3ml / min . Elution peaks corresponding to PS-TT, PS-P2K-TT and PS-P5K-TT were collected separately.

[0040] The purified product was identified by Superdex200 gel filtration column (1.0cm×30cm), the eluent was 20mM phosphate buffer (pH7.4), and the flow rate was 0.5ml / min. Such as figure 2 As shown, compared with the carrier protein, the peak time of PS-TT, PS-P2K-TT and PS-P5K-TT was significantly earlier. This indicates that the molecular weight of the carrier protein increases significantly after binding to the pneumonia capsular polysaccharide. Since the three combined products all elute in the outer water volume of the gel filtration column, the peak eluting times of...

Embodiment 3

[0042] Example 3: Determination of the immunogenicity of polysaccharide-conjugated vaccines

[0043] Select 32 5-week-old female Blab / C mice weighing 15-22 g. They were randomly divided into 4 groups, namely capsular polysaccharide group, PS-TT group, PS-P2K-TT group and PS-P5K-TT group, with 8 mice in each group. Intraperitoneal injection, each injection containing 5 micrograms of polysaccharides, once a week, a total of 3 injections. After 21 days, blood was collected from the orbit. Anti-capsular polysaccharide IgG, IgG1 and IgG2a in mouse plasma were detected by ELISA.

[0044] Such as Figure 4 As shown, the capsular polysaccharide group produced weak IgG and IgG1 titers against the capsular polysaccharide, while IgG2a was not detected. The antibody titer increased significantly after the capsular polysaccharide was coupled with the carrier protein. Compared with the capsular polysaccharide group, the anti-capsular polysaccharide IgG and IgG1 antibody titers in the P...

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Abstract

The invention provides a preparation method of a novel polysaccharide conjugate vaccine treating a PEG heterobifunctional reagent as a conjugation bridge. A meningococcal polysaccharide conjugate vaccine is exploited through the method. The method has the following advantages: 1, the respective self-crosslinking of polysaccharides and proteins in traditional methods is avoided, the yield is improved, and the quality control is benefited; and 2, a long PEG chain can increase the distance between the polysaccharides and the proteins, reduce the mutual space shield effect between the polysaccharides and the proteins, and improve the immunogenicity of the polysaccharide conjugate vaccine.

Description

technical field [0001] The invention provides a novel combination technology for preparing Neisseria meningitidis capsular polysaccharide conjugate vaccine; based on the combination technology, a meningitis capsular polysaccharide conjugate vaccine has been prepared, which can be used for the treatment of important diseases such as cerebrospinal meningitis Immunoprophylaxis belongs to the field of biomedicine. Background technique [0002] Meningitis is an epidemic of meningeal inflammation caused by Neisseria meningitidis, which has not yet been effectively controlled. Neisseria meningitidis is mainly transmitted by coughing, sneezing or kissing, invades the blood circulation from the nasopharynx, and finally is confined to the meninges and spinal cord membranes, forming purulent cerebrospinal meningopathy. Common symptoms include high fever, severe headache, and stiffness in the back of the neck. Drowsiness, vomiting, photophobia, or skin rash may also occur. The disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/095A61K47/48A61P31/04
Inventor 胡涛安文琪苏志国范蓓黄庆瑞马小伟潘若文
Owner 华兰生物疫苗股份有限公司
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