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Novel HPV therapeutic nucleic acid vaccine

A technology of HPV16-AVLC1 and HPV16-AVLS1, which is applied in the field of new HPV therapeutic nucleic acid vaccines, can solve the problems of weak immune response and limited therapeutic effect, and achieve the effect of efficiently inducing T cell immunity

Active Publication Date: 2022-03-04
BEIJING AEONVITAL BIOMEDICINE RES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

VLP as a subunit protein vaccine stimulates a weaker cellular immune response and thus exhibits limited therapeutic efficacy

Method used

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  • Novel HPV therapeutic nucleic acid vaccine
  • Novel HPV therapeutic nucleic acid vaccine
  • Novel HPV therapeutic nucleic acid vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Design scheme, construction and preparation of nucleic acid sequence

[0069] 1. Obtainment of HPV16 / 18 E6, E7, L1 and L2 target fragments

[0070] Download the DNA sequence and protein sequence of E6 / E7 and L1 / L2 of HPV16 strain NC_001526.4 from GenBank;

[0071] The DNA sequence and protein sequence of E6 / E7 and L1 / L2 of HPV18 strain AY262282.1 were downloaded from GenBank.

[0072] 2. Sequence optimization

[0073] After splicing all the sequences according to the vaccine protein structure, HPV16-AVLS1 and HPV16-AVLC1 contain 685 and 738 amino acids, respectively, and HPV18-AVLS1 and HPV18-AVLC1 contain 702 and 755 amino acids, respectively, and then optimize the codons. These optimization methods Including but not limited to: human codon usage bias, moderate GC content, stable mRNA secondary structure, etc., eliminating repetitive sequences and cryptic splice sites and unwanted restriction enzyme sites, while preventing tRNA pools in cells of exhaustion...

Embodiment 2

[0077] Example 2 Recombinant plasmid digestion and expression identification

[0078] 1. Enzyme digestion identification

[0079] The plasmids of AVL0318-HPV16 / 18-AVLS1 / AVLC1 were amplified separately using Escherichia coli AVL-DH5α(SacB), and then purified using the endotoxin-free plasmid purification kit.

[0080] The specific operation method is as follows:

[0081] 1) Use AVL-DH5α(SacB) strain on LB medium with 6% sucrose, culture at 37°C for 24h.

[0082] 2) Use the plasmid extraction kit for plasmid extraction.

[0083] 3) Use HindIII and XhoI double digestion to verify the correctness of the plasmid, and sequence to verify the correct type of the sequence, such as image 3 shown.

[0084] 2. Expression Identification

[0085] Plasmid expression identification was performed using the HEK-293T cell line. HEK-293T cells were cultured in a 6-well plate, and 1.5 μg of the plasmid was transferred into the cells using lipofectamine2000. After 48 hours of transfection, th...

Embodiment 3

[0086] Example 3 Cellular Immunity Analysis

[0087] 3.1 Immunization, C57BL / 6J mice were subcutaneously administered 30 μg HPV16 DNA vaccines AVL0318-AVLS1 and AVL0318-AVLC1 (1:1) or 30 μg HPV18 DNA vaccines AVL0318-AVLS1 and AVL0318 in the pinna of each ear at 2-week intervals - AVLC1 (1:1) (plasmid dissolved in TE buffer, i.e. 15 μg (20 μL) / ear) immunized 3 times. Use 3-5 mice per group. The immune response was detected one week after the three immunizations. ELISPOT of mouse splenocytes was used to detect E6 and E7 specific cellular immune responses.

[0088] 3.2 ELISPOT detection

[0089] 3.2.1 Acquisition of splenocytes

[0090] Take about 500 μL of blood from the mice in a 1.5mL EP tube, let it stand at room temperature for about 1 hour, take the serum and store it at -80°C; the mice are killed by neck dislocation, soaked in alcohol for 5 minutes, then transferred to the ultra-clean bench, laparotomy, and the spleen is taken . The taken spleen was gently ground, d...

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Abstract

The nucleic acid sequence provided by the invention comprises an HPV16-AVLS1 sequence and an HPV16-AVLC1 sequence in a ratio of 1: 1; the ratio of the HPV18-AVLS1 sequence to the HPV18-AVLC1 sequence is 1: 1; the sequence AVLS1 and the sequence AVLC1 respectively comprise two E6 proteins which are connected in series, two L1 short peptides, two L2 short peptides, two E7 proteins which are connected in series, a PADRE sequence and an adjuvant sequence; the N end of the sequence AVLS1 is provided with a mouse IgK secretory peptide sequence; the N end of the sequence AVLC1 is provided with a ubiquitin sequence. The nucleic acid sequence provided by the invention not only can induce a high-titer anti-E6 / E7 antibody, but also can induce high-level functional cellular immunity, and shows excellent effects of preventing and treating HPV-related tumors.

Description

technical field [0001] The invention relates to immunotherapy and prevention of tumors, in particular to a novel HPV therapeutic nucleic acid vaccine. Background technique [0002] Approximately 20-25% of cancer cases worldwide are caused by infectious agents. Approximately 15% of all human cancers are associated with viral infections. Human papilloma virus (HPV) causes approximately 30% of all infectious agent-associated cancers and is associated with more than 95% of cervical cancers. In 2018, more than 300,000 women worldwide died of cervical cancer. This is the result of persistent high risk HPV (hrHPV) infection. [0003] HPV preventive vaccines are currently available on the market, and preventive vaccines can prevent persistent cervical infection by inducing type-specific neutralizing antibodies against the major capsid protein L1. However, they are not effective in the treatment of pre-existing HPV infection or precancerous lesions. In addition, existing cervica...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/70C12N15/90C12N15/66C12N15/55C12N15/54A61K39/12A61K39/39A61P31/20
CPCC07K14/005C12N15/70C12N15/902C12N9/22C12N9/1055A61K39/12A61K39/39A61P31/20C12Y204/0101C12N2710/20022C12N2710/20034C07K2319/00C07K2319/95C07K2319/40C07K2319/036A61K2039/53A61K2039/575A61K2039/57A61K2039/55516Y02A50/30C12N15/111C12N15/85C12N15/63A61K2039/572A61K2039/70A61P35/00C12N2310/20
Inventor 张秀军刘磊唐建明
Owner BEIJING AEONVITAL BIOMEDICINE RES CO LTD
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