Adenovirus bivalent vaccine

A technology of adenovirus and virus particles, applied in the field of virus immunology, can solve problems such as difficulty in production, achieve the effects of preventing infection, improving safety and application range, and improving replication ability

Inactive Publication Date: 2020-05-19
GUANGZHOU N BIOMED LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the replication-deficient Ad3, Ad4 and Ad7 that only knock out the E1 and E3 genes are difficult to produce in the vaccine production cell line 293 or PerC6.

Method used

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  • Adenovirus bivalent vaccine
  • Adenovirus bivalent vaccine
  • Adenovirus bivalent vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of Example 1 Replication-deficient Ad4 Vaccine

[0043] 1. Construction of the Ad4 genome circularization shuttle vector

[0044] 1. Construction of Ad4 genome circularization shuttle vector.

[0045]The Ad4 genome was used as a template for PCR amplification to obtain recombinant arms Ad4-L and Ad4-R.

[0046] Ad4-L primer sequence:

[0047] Ad4-L Fw, ATAGAATTCGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO. 1);

[0048] Ad4-L Rw, TTTACTAGTGTTTAAACGTAATCGAAACCTCCACGTAATGG (SEQ ID NO. 2).

[0049] PCR program: 95°C, 30 seconds; 62°C, 30 seconds; 72°C, 20 seconds; 25 cycles.

[0050] Ad4-R primer sequence:

[0051] Ad4-R Fw, ACTAGTAGCTGGATCCAAGCCTCGAGGCACTACAATG (SEQ ID NO. 3);

[0052] Ad4-R Rw, CCTGCCGTTCGACGATGCGATCGCCATCATCAATAATATACCTTATAGATGG (SEQ ID NO. 4).

[0053] PCR program: 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 80 seconds; 25 cycles.

[0054] The Ad4 genome circularization shuttle plasmid pT-Ad4(L+R) was obtained by ligation with pSIMPLE 19 (EcoRV) v...

Embodiment 2

[0132] Preparation of Example 2 Replication Deficient Ad7 Vaccine

[0133] 1. Circularization of the Ad7 genome

[0134] 1. Construction of the shuttle plasmid pT-Ad7(L+R) for circularizing the Ad7 genome.

[0135] Using the Ad7 genome as a template, the left arm (L-Ad7) and the right arm (R-Ad7) of the Ad7 genome were obtained by PCR.

[0136] L-Ad7 primer:

[0137] L-Ad7-F: ACTGCGATCGCCTCTCTATTTAATATACCTTATAGATGG (SEQ ID NO. 25);

[0138] L-Ad7-R: ACATGGATCCTCACTGAAGATAATCTCCTGTGG (SEQ ID NO. 26).

[0139] PCR conditions: 95°C, 3min; 95°C, 30s; 56°C, 30s; 72°C, 40s; cycles 30; 72°C, 5min;

[0140] R-Ad7 primer:

[0141] R-Ad7-F: AGCTGGATCCGAACCACCAGTAATATCATCAAAG (SEQ ID NO. 27);

[0142] R-Ad7-R: TGAGCGATCGCCTCTCTATATAATATACCTTATAGATGGAA (SEQ ID NO. 28).

[0143] PCR conditions: 95°C, 3min; 95°C, 30s; 56°C, 30s; 72°C, 1min; cycles 30; 72°C, 5min;

[0144] The PCR product and the T vector were ligated with three fragments using Exnase recombinase to obtain pT-Ad7(L+R...

Embodiment 3

[0221] Embodiment 3 Ad4 and Ad7 bivalent vaccine preparation

[0222] 1. Vaccine storage

[0223] The replication-defective Ad4 and Ad7 vaccines purified by cesium chloride density gradient force centrifugation were carried out until the concentration of Ad4 was 4×10 11 vp / ml, the concentration of Ad7 is 4×10 11 vp / ml, stored at -80°C.

[0224] 2. Immunogenicity evaluation of Ad4 and Ad7 bivalent vaccine in macaques

[0225] The immunogenicity evaluation scheme of Ad4 and Ad7 quadrivalent vaccine in macaques was designed, as shown in Table 1, and the immunogenicity of Ad4 and Ad7 bivalent vaccine was evaluated according to the designed immunization scheme. Ad4 and Ad7 bivalent vaccine (Ad4: 2×10 10 vp / ml, Ad7: 2×10 10 vp / ml).

[0226] Table 1

[0227]

[0228] Adult rhesus monkeys were selected and divided into 2 groups with 4 monkeys in each group. Intramuscular injection (arm) was adopted for immunization. The first group was immunized with Ad4 and Ad7 bivalent va...

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Abstract

The invention discloses an adenovirus bivalent vaccine. The vaccine comprises a replication-deficient human adenovirus type 4 and a replication-deficient human adenovirus type 7. E1 and E3 genes of the replication-deficient human adenovirus type 4 and the replication-deficient human adenovirus type 7 are deleted, and partial coding frames of the E4 gene are replaced by corresponding coding framesof E4 gene of human adenovirus type 5. The vaccine can effectively stimulate an organism to generate a humoral immune response and a cellular immune response, generate specific neutralizing antibodieswith high titer, and is used for preventing infection of pathogens.

Description

technical field [0001] The invention belongs to the technical field of virus immunology, and in particular relates to an adenovirus bivalent vaccine. Background technique [0002] Adenovirus (Adenovirus, Ad) is a double-stranded DNA virus with a genome length of about 35-40 kb. It is known that human adenoviruses are divided into 7 subgroups (A-G), including more than 50 serotypes (more than 90 genotypes), which mainly cause acute respiratory diseases (adenovirus B and C subgroups) and conjunctivitis after infection. (adenovirus subgroups B and D) and gastroenteritis (adenovirus subgroup F 41 and 42, G subgroup 52). Respiratory tract infections caused by adenoviruses are mostly caused by adenovirus types 3, 4, and 7. Ad4 and Ad7 mainly concentrated in army, school and other places where youths and adolescents gathered, and even resulted in the death of patients. However, there is no specific drug for the treatment of adenovirus infection, and only supportive treatment can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/235A61K39/295A61P31/20
CPCA61K39/12A61K2039/70A61P31/20C12N2710/10334
Inventor 陈凌杨臣臣刘晓琳
Owner GUANGZHOU N BIOMED LTD
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