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HPV (human papillomavirus) peptide/DC (dendritic cell) mixed vaccine and preparation thereof

A hybrid vaccine, BM-DC technology, applied in the field of medical bioengineering, can solve the problems of clinical trials failing to achieve expected results, lack of anti-HPV drugs, limited induction and activation, etc.

Inactive Publication Date: 2011-04-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, most HPV therapeutic vaccines have limited induction and activation of human cellular immunity, and clinical trials have not achieved the expected results.
Since the immunity of patients with HPV infection is often relatively low, it is not enough to stimulate effective cellular immunity to clear the virus infection, and there is a lack of efficient and specific anti-HPV drugs in clinical practice. Therefore, how to enhance the immunogenicity of HPV protein and promote antigenic Improving the body's specific antiviral immunity by presenting and finding efficient adjuvants is a key issue that needs to be solved urgently in the research of HPV vaccines

Method used

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  • HPV (human papillomavirus) peptide/DC (dendritic cell) mixed vaccine and preparation thereof
  • HPV (human papillomavirus) peptide/DC (dendritic cell) mixed vaccine and preparation thereof
  • HPV (human papillomavirus) peptide/DC (dendritic cell) mixed vaccine and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of HPV11E7 polypeptide / DC mixed vaccine derived from mouse bone marrow

[0022] 1) Method:

[0023] The bone marrow of the mouse tibia and femur was blown into a cell suspension, and ammonium chloride was added to lyse the red blood cells. RPMI1640 culture solution 3ml / well was resuspended and cultured in six-well plate. After 48 hours, all the culture solution was discarded, and 2ml / well culture solution and cytokines were added. After 48 hours, all the cells were collected, resuspended and divided into 5 groups, each of which was incubated with different stimulants for 48 hours: (1) peptide (20 μg / ml) and imiquimod (2 μg / ml), (2) peptide (20μg / ml) and PIC (20μg / ml), (3) peptide (20μg / ml) and CpG (20μg / ml), (4) peptide (20μg / ml), (5)) blank control (without any Components), wherein the TLR ligands were added 2 hours after the addition of the peptide.

[0024] 2) DC morphology observation:

[0025] On the third day of DC isolation and culture,...

Embodiment 2

[0026] Example 2 Identification of DC surface molecular marker changes detected by flow cytometry:

[0027] 1) Method:

[0028] On day 7 of DC culture, DC (1×10 5 Each / 100ul) were co-incubated with FITC-labeled mouse CD40, CD80, CD11c and PE-labeled CD86, Iab antibodies for 30 minutes, and then flow cytometry detection was performed.

[0029] 2) Results:

[0030] By flow cytometry analysis, CD11c positive cells accounted for 81.3%. DC phenotypes were analyzed by flow cytometry on the 5th and 7th day, the results can be found in figure 1 , figure 1 After BM-DCs were co-incubated with HPV11E7 polypeptide and TLR agonist for 48 hours, DC surface markers CD40, CD80, CD86 and MHCII (I-Ak) were detected by flow cytometry to determine their maturity.

[0031] The results indicated that after only shocking with antigenic peptides, the maturity of DCs was slightly higher than that of the non-shocking group, but after adding TLR ligands, the maturity of DCs in each treatment group ...

Embodiment 3

[0035] Example 3 Inoculation of mice and observation of immunological characteristics

[0036] 1) Inoculation method:

[0037] After 5 groups of DCs with different treatments were washed at least twice with PBS, the cell concentration was adjusted to 3×10 5 / 200μl / mouse, injected into the peritoneal cavity of mice (group of 6×5). After that, inject once every other week for a total of 3 injections.

[0038] 2) Immunotherapy model protocol for tumor-bearing mice (in vivo experiment):

[0039] The mice were immunized with DC three times, and on the second day after the third immunization, B16 cells (1×10 5 / 200μl / piece). On the 19th day after tumor inoculation, all the mice were sacrificed, and splenocyte suspension was prepared. After 2 hours of attachment, the suspended cells were collected, and RPMI1640 culture medium containing muring IL-2 (20U / ml) and 10% fetal bovine serum After resuspension, culture them in a six-well plate (about 7×106 / ml), and add peptide (20 μg / ml...

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Abstract

The invention provides an HPV (human papillomavirus) peptide / DC (dendritic cell) mixed vaccine prepared in the presence of a TLR (toll-like receptor) agonist. The mixed vaccine comprises one part of HPV11E77-15, one part of mouse bone marrow-derived dendritic cells which are cultured in vitro and one part of TLR9 agonist as an adjuvant, wherein the dendritic cells are extracted from mouse bone marrow and further cultured in vitro, the co-incubation with one section of HPV11E7CTL (cytotoxic T lymphocyte) epitope peptide with strongest immunogenicity is firstly carried out, the co-incubation with the TLR ligand CpG and the like is further respectively carried out for promoting the maturation of the DC, the degree of maturation can be confirmed by detecting the change of a marker on the surface of the DC after incubation, and the DC vaccine which is simulated to mature can be used for immunizing mice. The TLR ligand and the HPV11E7 peptide are utilized jointly to promote the degree of maturation of the DC, the level of TNF-alpha (tumor necrosis factor-alpha) and IFN-gamma (immunoreactive fibronectin-gamma) of factors of secretory cells of T cells can be particularly improved after combination of the CpG, and the HPV peptide / DC mixed vaccine can be used for preventing and treating genital warts and cervical cancer.

Description

technical field [0001] The invention belongs to the technical field of medical bioengineering, and in particular relates to an agonist of Toll-like receptor (TLR), which is used as an adjuvant of HPV11E7 epitope peptide-specific DC vaccine, and is expected to be used for the prevention and treatment of condyloma acuminatum and cervical cancer. Background technique [0002] Human papillomavirus (HPV) can cause the current high incidence of sexually transmitted diseases in my country - condyloma acuminatum and HPV highly related cervical cancer. The pathogens of condyloma acuminatum are mainly HPV6 and 11 types, with a high recurrence rate and stubborn intractability; while cervical cancer has a poor curative effect and prognosis, and is showing a younger trend year by year, both of which have become serious threats to patients, families, and even society. Hidden danger. [0003] Although cervical cancer preventive vaccines have been marketed in many countries, and have shown...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/39A61P31/20A61P35/00
Inventor 茅晓红陈贤祯张巍巍王建有刘伦飞周强朱可建程浩
Owner ZHEJIANG UNIV
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