CN110734477A

CN110734477A - Papillomavirus related antigen short peptide and application thereof

Info

Publication number: CN110734477A
Application number: CN201911128812.3A
Authority: CN
Inventors: 黄燕花; 赵乙木; 罗夫·辛克纳吉; 孙晨
Original assignee: Vitan Guangzhou Pharmaceutical Co Ltd
Current assignee: Vitan Guangzhou Pharmaceutical Co Ltd
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2020-01-31

Abstract

The invention discloses a papilloma virus related antigen short peptide and application thereof, wherein the sequence of the papilloma virus related antigen short peptide is shown as SEQ ID NO: 1-16- , the short peptide has high affinity with MHC class I and MHC class II molecules on DC cells, can effectively play a role in antigen presentation, and has good potential of a polypeptide vaccine and a DC vaccine, at least in the short peptide sensitizes dendritic cells in vitro, and the prepared DC vaccine can prevent papilloma virus related diseases and has good clinical transformation prospect.

Description

Papillomavirus related antigen short peptide and application thereof

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, it relates to papillomavirus-associated antigen short peptides and uses thereof.

Background

Human Papilloma Virus (HPV). HPV mainly causes proliferative lesions of human skin and mucous membrane, the HPV genome is about 7.9KB, and the stated virus particle diameter is 52-55 nm. Has high affinity for skin mucosa epithelial cells, and can cause spine cell hyperplasia, course epidermal thickening and epidermal keratinization. It is proved that HPV can be involved in the occurrence of various tumors, and the infection rate of HPV16, HPV18 and HPV33 in patients with ovarian cancer is higher; elevated gene and antibody expression of HPV16 and HPV18 in lung cancer patients; HPV is also a risk factor for esophageal cancer, which can cause malignant transformation and carcinogenesis of esophageal epithelial cells; HPV16, HPV18 expression is elevated in colorectal cancer tissue. In addition, HPV can also be involved in the progression of various chronic diseases, such as chronic obstructive pulmonary disease and the like.

HPV has an effective vaccine at present, and can play a role in preventing cervical cancer. There is still a great need for the exploration of more and more optimal HPV vaccines. Immunology has shown that Dendritic Cells (DC) are the most powerful antigen-presenting cells known at present, and are considered to be the initiator of the immune response of the body, and are centrally located in the immune response. Mature DCs express rich, antigen-presenting-associated MHC class I and MHC class II molecules that can ingest, process, and present antigen; participate in the maintenance of immunological memory; secretion of cytokines modulates immune responses. Papillomavirus antigen polypeptides that bind to MHC class I and MHC class II molecules are particularly critical.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the existing papillomavirus vaccine, provide a papillomavirus antigen polypeptide which can be combined with MHC class I and MHC class II molecules, provide an HPV vaccine based on a DC technology, be applied to preventing and treating HPV related chronic diseases by adopting the DC technology, increase the activity of mature DC cells by combining stem cell factors/vitrogen, promote the DC cell activation through a plurality of HPV specific antigen peptides, achieve the purpose of preventing papillomavirus infection and treating, and have good application prospect.

The invention aims to provide HPV antigen-associated short peptides.

The invention also aims to to provide the application of the HPV antigen-associated short peptide in the preparation of HPV antigen polypeptide DC vaccines.

The invention further aims to to provide HPV antigen polypeptide DC vaccines.

The above purpose of the invention is realized by the following technical scheme:

provides HPV related antigen short peptide, the sequence of which is shown in SEQ ID NO 1-16 any .

Provides the application of the HPV related antigen short peptide in the preparation of papillomavirus antigen polypeptide DC vaccines.

Provides the application of the HPV related antigen short peptide in preparing the medicine for preventing and treating HPV related diseases.

The DC vaccine for preventing and treating papilloma virus related lentidiseases is obtained by loading the papilloma virus related antigen short peptides and dendritic cells, and specifically, the autologous dendritic cell preparation is obtained by combining short peptides shown by at least in SEQ ID NO. 1-16 to sensitize the dendritic cells in vitro.

The papillomavirus related antigen short peptide of SEQ ID NO. 1-SEQ ID NO. 16 can induce DC to play the role of antigen presentation.

In particular, the vaccine is an intravenous infusion vaccine.

In the preparation method of the DC vaccine for preventing and treating the papillomavirus related slow disease, the stem cell factor or the vitrogen factor is selected as an adjuvant to increase the activity of the DC cell, and the specific antigen peptide is used for sensitizing dendritic cells in vitro to obtain an autologous dendritic cell preparation which is used as the intravenous infusion type papillomavirus related slow disease prevention and treatment vaccine. The preparation method comprises the following steps: the maturation of the DC cells is promoted by taking a maturation promoting factor and simultaneously taking a stem cell factor or a Vitrogen factor as an adjuvant; then adding the short peptide of claim 1 into a DC cell culture system for inducing maturation, collecting the DC cells loaded with the short peptide fragment, washing with physiological saline, and then resuspending with physiological saline to obtain the DC vaccine.

More specifically, as an alternative, the DC vaccine for papillomavirus-related lentigo prevention and treatment is prepared by the following steps:

s1, extracting and inducing DC cells:

s11, obtaining immature DC cells

Collecting peripheral blood of healthy donor, separating mononuclear cells by lymphocyte separation, culturing at 37 deg.C in culture medium with 5% CO₂After culturing for 3 hours under the conventional condition, the adherent cells are immature DC cells;

s12, amplification culture of immature DC cells

37℃、5％CO₂Culturing for 5 days under the condition, and changing the culture solution every other day to complete the amplification culture of immature DC cells (imDC cells);

S13.Induction of DC cells

Adding a maturation promoting factor, and simultaneously taking a stem cell factor or a Vitrogen factor as an adjuvant to promote the maturation of the DC cells;

s2, loading of polypeptide:

adding the short peptide of claim 1 to the culture system 5 days after inducing DC cell maturation;

s3, preparing a DC vaccine:

and (3) centrifuging to collect the DC cells loaded with the short peptide fragments, washing the cells for 3 times by using physiological saline, and finally, resuspending the DC cells loaded with the short peptide fragments by using the physiological saline to obtain the DC vaccine.

In addition, the application of the DC vaccine in preparing the prevention and treatment medicines for the papillomavirus related chronic diseases also falls into the protection scope of the invention.

The invention predicts that HPV can be used as a sequence cluster of an antigen by combining with bioinformatics technology, and after a large number of researches and searches, the obtained 16 papillomavirus antigen peptides have high affinity with MHC class I and MHC class II molecules on DC cells, can effectively play a role in antigen presentation, have the potential of good polypeptide vaccines and DC vaccines, and prompt that the HPV antigen peptides have good clinical transformation and disease prevention and treatment prospects.

The invention selects specific epitope polypeptide, sensitizes autologous DC cells in vitro, prepares DC cell preparation, and carries out venous return transfusion on patients, reconstructs the whole body immune balance of organisms, starts immune system, and prevents the specificity of infected microorganisms.

The DC technology adopted by the invention jointly activates dendritic cells by using a plurality of specific antigen peptides, the antigen peptides have extremely strong specificity, induce dendritic cells with higher activity to carry a plurality of antigen information, can stimulate the immunity of an organism after being infused back into a human body, can achieve the aim of inducing the human body to generate specific antibodies aiming at papilloma virus and CTL cells aiming at the specificity of the papilloma virus, and thus can effectively prevent and treat the occurrence and development of chronic diseases related to the papilloma virus.

The invention has the following beneficial effects:

the invention provides papilloma virus antigen polypeptides with sequences shown as SEQ ID NO. 1-16, which have high affinity with MHC class I and MHC class II molecules on DC cells, can effectively play a role in antigen presentation, and have good potential of polypeptide vaccines and DC vaccines.

The short peptide has short length and small chemical synthesis difficulty, can be directly synthesized to obtain a high-purity product, greatly reduces the application cost, has good potential of a polypeptide vaccine and a DC vaccine, can prevent related diseases of papilloma virus, particularly related chronic diseases, and has good clinical transformation and practical application prospects.

The technology for preparing the DC vaccine for preventing and treating the papilloma virus based on the papilloma virus antigen peptide has a plurality of advantages: (1) after DC is activated in vitro, the stem cell factor/Vitrogen factor can increase the activity of DC cells, increase the antigen-recognizing ability, and promote the generation of immune response after being returned into the body. (2) Long-term immunological memory: because the specific antigen peptide is fully contacted with the immune cells with memory function, the immune cells have precise and long-term immune memory after being infused back into the body, the immune control effect is enhanced, and long-term protection is provided for preventing reinfection or preventing. (3) After the DC is back-transfused in vivo, the immunity of the organism can be reestablished, thereby achieving the purpose of preventing and treating the in vivo infection virus.

Drawings

FIG. 1 is a comparison of killing activity against target cells in the DC vaccine treated group and the polypeptide-DC vaccine control group.

FIG. 2 shows the activity change of T cells in a blank control group, a DC vaccine group, a polypeptide vaccine group and a polypeptide-DC vaccine group detected by a CCK-8 method in vitro DC sensitized T cells.

FIG. 3 shows the levels of antibodies in sera of the placebo, DC, polypeptide, and polypeptide-DC vaccine groups measured by ELISA 6 weeks after immunization of mice.

Detailed Description

The invention is further illustrated at in the accompanying drawings and detailed description, which are not intended to limit the invention in any way, except as otherwise indicated, reagents, methods and equipment used in the practice of the invention are conventional in the art and reagents and materials are commercially available in the following examples.

Example 1

Dendritic Cell therapy technology (Dendritic Cell, DC) is that after mononuclear cells of autologous peripheral blood are collected, specific Cell factors are added under strict aseptic environment, meanwhile, stem Cell factors/Vitrogen factors are supplemented, transformed more active Dendritic cells (Dendritic cells, DC) with antigen presenting function are obtained, HPV antigen polypeptide is added in days before feedback, the DC obtains mature Dendritic cells sensitized by high-risk pathogenic virus specific antigen peptide by recognizing and capturing various single epitope antigen peptides, and the Dendritic cells can generate a large amount of antibodies generated aiming at specific antigens by self by utilizing the targetable migration and immunocompetence of the Dendritic cells, so that the body immune balance is reconstructed, and the prevention of pathogenic microorganisms is more directional and more active by preventing and treating pathogenic virus infection, and the occurrence of papilloma virus related lenti diseases is fundamentally prevented and treated.

The objective of this study was to develop and screen papillomavirus antigen polypeptides that bind to MHC class I and MHC class II molecules for the preparation of DC technology-based papillomavirus vaccines.

1. Prediction of HPV antigen binding ability to MHC class I and MHC class II molecules polypeptides:

determining HPV sequence clusters (https:// www.uniprot.org/uniref /) through a uniprot database, inputting the sequence clusters (http:// www.ddg-pharmac. net/VaxiJen. html) for online analysis, setting Threshold to be 0.6, determining HPV antigen protein, predicting short peptide sites with high affinity of antigen and MHC class I and MHC class II molecules through an IEDB database, and obtaining series of polypeptides.

2. And (3) screening 50 polypeptide fragments with high prediction scores and low prediction toxicity from the polypeptides, and performing -step research.

(1) DC cell expansion and maturation

Collecting peripheral blood 50ml of healthy donor, separating mononuclear cells by lymphocyte separation, culturing in culture medium (purchased from Gibico Co.) at 37 deg.C under 5% CO₂After 3 hours of conditions, adherent cells were immature DCs. Immature DC, 37 ℃ 5% CO₂And (5) culturing for 5 days (changing liquid every other day), and completing the amplification culture of the imDCs. Maturation factors are added, and stem cell factors or Vitrogen factors are used as adjuvants to promote DC maturation.

(2) Killing activity of specific CTL against target cells

The killing activity assay was performed using polypeptide-DCs-induced activated T lymphocytes as effector cells and positive fibroblasts expressing HPV as target cells.

The method is divided into four groups: the polypeptide-DCs in the treatment group induce activated T cells, and the DCs in the control group induce activated T cells, according to the ratio of the number of effector cells to target cells of 2: 1 adding into 96-well culture plate, repeating 3 wells each, arranging effector cell group and target cell group as blank control, and standing at 37 deg.C and 5% CO₂Culturing in an incubator for 12h, detecting the OD 450nm values of the experimental wells and the control wells by a CCK-8 method, and calculating the killing rate.

Killing rate [ target cell OD value + effector cell OD value-experimental well OD value ]/target cell OD value

The results show that the best polypeptide is selected according to the inhibition of polypeptide-DCs-induced activated CTL on target cells, the corresponding relation is not obviously related with the predicted prediction scores of 50 polypeptides, and the reference of the prediction result is poor.

The sequences of the best 16 finally selected polypeptides are shown in Table 1, the inhibitory data of the 16 polypeptide-DCs induced activated CTL on target cells are shown in figure 1, and the inhibitory activity of the polypeptide-DCs induced activated CTL on the target cells is obviously higher than that of the polypeptide-unloaded DCs induced activated CTL.

TABLE 1

(3) Induced activation of T lymphocytes

of the 16 polypeptides were randomly extracted, the obtained polypeptide-DCs were added with mitogen and incubated to lose their proliferative activity, washed 2 times with PBS, mixed in 96-well culture plates at a concentration ratio of DC: T of 1:20 and co-cultured, divided into four groups, negative control group, polypeptide group, DC group, and polypeptide-DC group, the solution was changed every 2 days, equal amounts of polypeptide-DCs were added until the fourth day of culture, activation and proliferation of specific CTL clone was induced by repeated stimulation, and cell activity was detected until day 6, as shown in FIG. 2, the polypeptide-DC group was found to be the strongest in activity.

3. Immunogenicity of HPV-specific DC vaccines

Immunogenicity was tested by randomly drawing out of the above 16 polypeptides.

Each group of 5 BALB/C female mice, 6-8 weeks old, divided into 4 groups, polypeptide vaccine, polypeptide-DC vaccine, DC vaccine (negative control), and physiological saline as blank control, were injected intramuscularly to mice 2X 10⁵Cells/mouse, were immunized twice in total, 2 weeks apart. After 6 weeks of immunization, the eyes were removed and blood was collected, and serum was separated (stored at-80 ℃ C.) for immunoassay.

The serum anti-HPV IgG antibody level was detected by ELISA kit, the results are shown in FIG. 3, both the DC vaccine group and blank group were negative; the polypeptide-DC group is 80%, and the serum of the polypeptide vaccine group mouse is 40% positive. And the antibody titer of the polypeptide-DC group is obviously higher than that of the pure polypeptide vaccine group. The result shows that the polypeptide-DC vaccine developed by the invention has higher positive conversion number.

The experiments show that the DC vaccine prepared by the invention can effectively play the role of antigen presentation, induce the organism to generate specific cellular immune response and humoral immune response of HPV antigen, and thus is expected to achieve the curative effect of preventing and treating HPV-related chronic diseases.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

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Claims

1. The papillomavirus related antigen short peptide is characterized in that the sequence is shown as SEQ ID NO:1-16 or .

2. Use of the short peptide of claim 1 for the preparation of a DC vaccine for the prevention and treatment of papillomavirus-related lenti-diseases.

3. The use of the short peptide of claim 1 for the manufacture of a medicament for the prevention or treatment of chronic diseases associated with papillomavirus.

papillomavirus antigen polypeptide DC vaccine, characterized by being loaded with the short peptides and dendritic cells according to claim 1.

5. The DC vaccine according to claim 4, wherein the DC vaccine is an autologous dendritic cell preparation obtained by in vitro priming of dendritic cells with a short peptide represented by at least of SEQ ID NOS: 1 to 16.

6. The DC vaccine of claim 4, wherein the vaccine is an intravenous infusion vaccine.

7. The DC vaccine of claim 4, wherein the activity of mature DC cells is increased by using stem cell factor or vitrogan factor as adjuvant.

8. The DC vaccine of claim 4, which is prepared by the following steps: the maturation of the DC cells is promoted by taking a maturation promoting factor and simultaneously taking a stem cell factor or a Vitrogen factor as an adjuvant; then adding the short peptide of claim 1 into a DC cell culture system for inducing maturation, collecting the DC cells loaded with the short peptide fragment, washing with physiological saline, and then resuspending with physiological saline to obtain the DC vaccine.

9. Use of DC vaccine according to any one of claims 4 to 8 for the manufacture of a medicament for the prevention and treatment of chronic disease associated with papillomavirus.