Genetically engineered vaccine against both Enterovirus 71 and Coxsackie virus A16

An amino acid, antigen epitope technology, applied in the field of biomedicine, can solve the problem of CA16 without cross-protection

Inactive Publication Date: 2015-11-25
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the development of HFMD vaccines is mainly aimed at EV71, neither the inactivated EV71 whol

Method used

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  • Genetically engineered vaccine against both Enterovirus 71 and Coxsackie virus A16
  • Genetically engineered vaccine against both Enterovirus 71 and Coxsackie virus A16
  • Genetically engineered vaccine against both Enterovirus 71 and Coxsackie virus A16

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1H

[0122] Expression and purification of embodiment 1 HBcPEP71 fusion protein

[0123] The 77th-83rd amino acid of HBc molecule is located at the top of its α-helical hairpin structure, which is the part that forms the spike on the surface of HBc virus-like particles, and is also the main immunodominant region (Major ImmunodominantRegion, MIR) of HBc protein. Such as figure 1 As shown in -A, amino acids 77-83 in the MIR region of the HBc molecule were replaced with the neutralizing epitope PEP71 (FGEHLQANDLDYGQ; SEQ ID NO.: 1) of CA16 to obtain a recombinant plasmid expressing the fusion protein HBcPEP71, and the sequence was confirmed to be correct by sequencing.

[0124] The amino acid sequence of Hbc is:

[0125] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNVGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRTPSPPRRRRSPSPRRRRSQSRESQC (SEQ ID NO.: 2)

[0126] The amino acid sequence of HBcPEP71 is:

[0127] MDIDP...

Embodiment 2

[0132] Example 2 Assembly of HBcPEP71 fusion protein into chimeric virus-like particles

[0133] In order to identify the assembly of HBcPEP71, the purified protein was layered on the top layer of a 10-50% sucrose density gradient for ultracentrifugation, and then 10 fractions were taken from top to bottom for Western Blot detection with HBc-specific monoclonal antibody. Such as image 3 As shown in -A, consistent with unmodified HBc, the HBcPEP71 fusion protein signal has specific signals in the last few components, and the signal of the seventh component is the strongest, indicating that HBcPEP71 exists in the form of particles. In order to detect the display of epitope PEP71 on the surface of HBcPEP71 particles, each component was detected by ELISA with PEP71-specific serum, and it was found that HBcPEP71 could be well recognized by PEP71-specific serum, while HBc could not ( image 3 -B), and consistent with the results of WesternBlot, the signal of the 7th component is t...

Embodiment 3

[0135] Example 3 HBcPEP71 immunized mouse serum can neutralize CA16

[0136] Three groups (6 / group) of 6-8 week-old female ICR mice were used to detect the immunogenicity of chimeric VLPs. The mice in each group were injected intraperitoneally with aluminum adjuvant-adsorbed PBS, HBc or HBcPEP71, in which HBc and PBS The group is the control. The neutralizing activity of the mixed sera of mice in each group two weeks after the fourth immunization was measured against CA16 / SZ05, and it was found that the immune sera of the PBS and HBc groups had no neutralizing activity when diluted 1:8, while the neutralizing activity of the immune sera of the HBcPEP71 group was and a titer of 16 (Table 1). The neutralizing activity of a single mouse serum to CA16 / SZ05 was further determined, and the results showed that the serum of 2 of the 6 HBcPEP71-immunized mice was not neutralized at 1 / 8 dilution, and the neutralizing titer of the other four mice was the lowest 8, up to 64 ( Figure 4...

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Abstract

The invention provides an epitope peptide. The epitope peptide is originated from the neutralizing epitope PEP71 of the Coxsackie virus and has a length of 5 to 15 amino acids; and recombinant protein formed by fusion between the epitope peptide and carrier protein can induce immunoreaction of a mammal to the epitope peptid. Experiments prove that the fusion protein including the epitope peptide can be used as a genetically engineered vaccine against both EV 71 and CA16.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to a genetically engineered vaccine against enterovirus 71 and coxsackie virus A16 and its application. Background technique [0002] Hand, foot and mouth disease (HFMD) is a common disease prevalent in children under 5 years old, enterovirus 71 (EV71) and Coxsackievirus A16 (Coxsackievirus A16, CA16) are the main causes of HFMD. Pathogens, both belong to the genus Human Enterovirus in the Picornaviridae family. Epidemiological surveys show that EV71 infection is the main cause of severe illness and death in HFMD. CA16 infection generally leads to mild symptoms, such as fever, mouth ulcers, herpes on hands and feet, etc. However, epidemiological surveys in recent years have shown that the number of severe neurological symptoms and death caused by CA16 infection is increasing. In addition, the current co-prevalence of EV71 and CA16 viruses has caused some patients ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K7/06C07K19/00C12N15/62A61K38/10A61K38/08A61K39/125A61P31/14G01N33/68
Inventor 黄忠叶晓华
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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