Click-chemistry-based pneumococcal polysaccharide conjugate vaccine and preparation method thereof

A technology combining vaccines and pneumonia, applied in the field of biomedicine, can solve the problems of affecting the structural stability of polysaccharides and proteins, affecting the immunogenicity, and long reaction time, so as to reduce the space shielding effect, improve the immunogenicity, and increase the space. effect of distance

Active Publication Date: 2013-05-22
华兰生物疫苗股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this combination technology has the following disadvantages: (1) the reaction time between the aldehyde group produced by the oxidized polysaccharide and the amino group of the carrier protein is too long, it takes more than 5 days, and the efficiency of the combination reaction is low; (2) some polysaccharides The reaction with carrier protein needs to be carried out at a higher temperature (such as 37°C), which will affect the structural stability of polysaccharides and proteins; (3) The immunogenicity of polysaccharide-protein conjugate vaccines needs to be further improved; polysaccharides and carriers The linking bridge of the protein is a methylamino group (-CH 2 -NH-), because the length of the methylamino group is too short, the steric shielding effect of the carrier protein will cover the antigenic epitope of the polysaccharide molecule, thereby affecting its immunogenicity
This combination technology has the following disadvantages: (1) The reactivity of cyanogen bromide is too strong, and the activation reaction is not easy to control, resulting in poor batch stability of the preparation process; (2) EDC mediates the combination of ADH-derived polysaccharides and carrier proteins At the same time, it is easy to cause self-crosslinking of polysaccharides and carrier proteins
These deficiencies can affect the immunogenicity and stability of polysaccharide conjugate vaccines

Method used

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  • Click-chemistry-based pneumococcal polysaccharide conjugate vaccine and preparation method thereof
  • Click-chemistry-based pneumococcal polysaccharide conjugate vaccine and preparation method thereof
  • Click-chemistry-based pneumococcal polysaccharide conjugate vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of short-chain pneumonia capsular polysaccharide conjugate vaccine (S-PCV)

[0032] The preparation reaction of short-chain pneumonia capsular polysaccharide conjugate vaccine (S-PCV) is as follows: figure 1 shown. 5mg of pneumonia capsular polysaccharide was dissolved in 50mM sodium acetate buffer (pH 4.7), and hydrolyzed at 75°C for 30 minutes. The pH value of the hydrolyzate was adjusted to 5.8, and reacted in the presence of sodium periodate at room temperature and protected from light for 10 minutes. Dialysis removes unreacted sodium periodate. The pH of the pretreated pneumonia capsular polysaccharide solution was adjusted to 7.4, and in the presence of sodium cyanoborohydride, the mass ratio of polysaccharide and aminoacetylene was mixed and reacted at a ratio of 2:1, and reacted at 37°C for 6 hours. Centrifuge 3 times at 5000 g using a filter membrane with a molecular weight cut-off of 50 kDa to remove unreacted sodium cyanoborohydride ...

Embodiment 2

[0034] Example 2: Preparation of long-chain pneumonia capsular polysaccharide conjugate vaccine (L-PCV)

[0035] The preparation reaction of long-chain pneumonia capsular polysaccharide conjugate vaccine (L-PCV) is as follows: figure 2 shown. 5mg of pneumonia capsular polysaccharide was dissolved in 50mM sodium acetate buffer (pH 4.7), and hydrolyzed at 75°C for 30 minutes. The pH value of the hydrolyzate was adjusted to 5.8, and reacted in the presence of sodium periodate at room temperature and protected from light for 10 minutes. Dialysis removes unreacted sodium periodate. The pH of the pretreated pneumonia capsular polysaccharide solution was adjusted to 7.4, and in the presence of sodium cyanoborohydride, the mass ratio of polysaccharide to aminoacetylene was mixed and reacted at a ratio of 2:1, and reacted at 37°C for 6 hours. Centrifuge 3 times at 5000 g using a filter membrane with a molecular weight cut-off of 50 kDa to remove unreacted sodium cyanoborohydride an...

Embodiment 3

[0037] Example 3: Purification and Characterization of L-PCV and S-PCV

[0038] The binding products involved in Examples 1 and 2 were 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 L-PCV and S-PCV were collected separately.

[0039] 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 image 3 As shown, compared with the carrier protein, the peak time of L-PCV and S-PCV was significantly earlier. This indicates that the molecular weight of the carrier protein increases significantly after binding to the pneumonia capsular polysaccharide.

[0040] use 1 H-NMR was performed to detect L-PCV and S-PCV. Such as Figure 4 As shown, compared with pneumonia polysaccharide molecules, S-PCV has protein characteristic peaks...

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Abstract

The invention relates to a polysaccharide conjugate vaccine for preventing pneumonia streptococcal infection, which is prepared by connecting pneumococcal capsular polysaccharide to tetanus toxoid by using a 'click chemistry' method. The immunogenicity of the polysaccharide conjugate vaccine is improved by prolonging the length of a connecting bridge between the pneumococcal capsular polysaccharide and the tetanus toxoid.

Description

field of invention [0001] The invention relates to a vaccine drug in the field of biomedicine, in particular to a pneumonia polysaccharide conjugate vaccine; the invention also relates to its preparation method and immunological evaluation. Background technique [0002] Streptococcus pneumoniae is a Gram-positive sac-shaped diplococcus that can cause noninvasive infections such as sinusitis, otitis media, tracheitis, and pneumonia, as well as invasive infections such as sepsis and meningitis. Streptococcus pneumoniae can be transmitted through the respiratory secretions of patients and carriers, and is one of the main causes of morbidity and death in infants, children and the elderly. Despite the availability of effective antibiotic treatment, respiratory disease and invasive infections caused by this bacteria still constitute a major public health problem. The World Health Organization (WHO) estimates that millions of people die from pneumococcal infection every year. In ...

Claims

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Application Information

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IPC IPC(8): A61K39/385A61K39/09A61K47/48A61P31/04
Inventor 胡涛安文琪苏志国范蓓吴鼎龙马小伟潘若文
Owner 华兰生物疫苗股份有限公司
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