CN110698544A

CN110698544A - EB virus related antigen short peptide and application thereof

Info

Publication number: CN110698544A
Application number: CN201911127136.8A
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: 2020-01-17

Abstract

The invention discloses EB virus related antigen short peptide and application thereof. The EB virus related antigen short peptide sequence is shown in any one of 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 polypeptide vaccines and DC vaccines. At least one in-vitro sensitized dendritic cell in the short peptide can be used for preparing the DC vaccine which can prevent related diseases of the EB virus and has good clinical transformation prospect. Moreover, the antigen 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 definite effect and has good application prospect.

Description

EB virus related antigen short peptide and application thereof

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, relates to EB virus related antigen short peptide and application thereof.

Background

Epstein-Barr virus (EBV) is a member of the genus lymphotropic virus of the family Herpesviridae, is the causative agent of infectious mononucleosis, has the biological property of specifically infecting human and certain primate B cells in vitro and in vivo, and is transmitted mainly through saliva. EBV is similar to other herpesviruses under an electron microscope, but has different antigenicity, the EBV genome is about 172KB, at least more than 100 proteins can be coded, B cells are main infection target cells of the EBV, and the EBV can directly enter a latent state after entering a human body and is characterized in that the virus is continuously present, the expression of virus proteins is limited, and the EBV has the potential of being gathered into a replication cycle.

EB virus has close correlation with occurrence of nasopharyngeal carcinoma and lymphoma of children, is listed as one of human tumor viruses which are possibly carcinogenic, and EB virus infection can promote development of blood tumor. In addition, it has been demonstrated that the EB virus antibody of Alzheimer's disease patient is obviously increased; the EB virus of the type 1 diabetes mellitus person causes stronger immune response; the EB virus expression of the breast cancer tissues is increased, and the EB virus gene expression and the antibody of the breast cancer patients are higher than those of the control group. May contribute to mammary epithelial cell malignancy by activating the HER2/HER3 signaling pathway; in patients with gastric cancer, EB virus promotes gene acetylation, and gene mutation participates in the canceration process.

Currently, antiviral drugs with positive curative effects on EBV infection are not available. For example, during acyclovir administration, EBV expulsion from the pharynx is reduced, but the symptoms of infectious mononucleosis are not improved, and EBV lymphoma treatment in immunodeficient patients is also ineffective. In recent years, advances have been made in the purification of EBV polypeptides, possibly by means of antibody or cellular immunity to block the primary infection of EBV. Its primary infection can promote the body to produce specific neutralizing antibody and cell immune response, and the cell immune and neutralizing antibody plays an important role in preventing exogenous reinfection and limiting primary infection and chronic infection. But their immunity is often not long lasting and strong.

The vaccine is the most effective method for preventing EBV infection, but the gene recombinant vaccine developed in China is under observation. 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. EB virus 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 EB virus vaccine, provide EB virus antigen polypeptide capable of being combined with MHC class I and MHCII molecules, provide an EBV vaccine based on DC technology, be applied to preventing and treating EBV related chronic diseases by adopting DC technology, increase the activity of mature DC cells by combining stem cell factor/vitrogen, promote the activation of DC cells through various EBV specific antigen peptides, achieve the purpose of preventing EB virus infection and treating, and have good application prospect.

The invention aims to provide an EBV antigen-associated short peptide.

The invention also aims to provide the application of the EBV antigen-associated short peptide in preparing the EBV antigen polypeptide DC vaccine.

The invention further aims to provide an EBV antigen polypeptide DC vaccine.

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

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

Provides the application of the EBV-associated antigen short peptide in the preparation of the EB virus antigen polypeptide DC vaccine.

Provides the application of the EBV-associated antigen short peptide in preparing the medicines for preventing and treating EBV-associated diseases.

In addition, the DC vaccine for preventing and treating the EB virus related lentivirus is obtained by loading the EB virus related antigen short peptide and dendritic cells. Specifically, the autologous dendritic cell preparation is obtained by sensitizing dendritic cells in vitro by combining short peptides shown by at least one of SEQ ID NO. 1-16.

The EB virus related antigen short peptide described by 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 EB virus related lentivirus, the stem cell factor or the vitrogen factor is selected as an adjuvant to increase the activity of DC cells, and specific antigen peptide is used for sensitizing dendritic cells in vitro to obtain an autologous dendritic cell preparation which is used as a venous return transfusion type EB virus related lentivirus preventing and treating 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 preventing and treating the EB virus related lentivirus 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 medicine for preventing and treating the EB virus related chronic diseases also belongs to the protection scope of the invention.

The invention predicts that the EBV can be used as a sequence cluster of the antigen by combining with a bioinformatics technology, and after a large number of researches and searches, the obtained 16 EB virus antigen peptides have high affinity with MHC I and MHC 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 EB virus 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 the dendritic cells with higher activity to carry a plurality of antigen information, can stimulate the immunity of an organism after being back-infused into a human body, can achieve the aim of inducing the human body to generate specific antibodies aiming at EB virus and CTL cells aiming at the specificity of the EB virus, and thus can effectively prevent and treat the occurrence and development of chronic diseases related to the EB virus.

The invention has the following beneficial effects:

the invention provides EB virus antigen peptides 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 EB virus related diseases, 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 EB virus based on the EB virus antigen peptide has a plurality of advantages that: (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 described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise specified; the reagents and materials used in the following examples are all commercially available.

Example 1

Dendritic Cell therapy (DC) is a technique of obtaining transformed, more active Dendritic Cells (DC) with antigen presenting function by collecting autologous peripheral blood mononuclear cells, adding specific cytokines under a strict aseptic environment, and simultaneously supplementing stem Cell factor/Vitrogen factor. The EB virus antigen polypeptide is added one day before feedback, the DC identifies and captures a plurality of single epitope antigen peptides to obtain mature dendritic cells sensitized by high-risk pathogenic virus specific antigen peptides, and the dendritic cells can be used for targeting migration and immunocompetence to induce a human body to generate a large amount of antibodies generated aiming at specific antigens, so that the immune balance of an organism is reconstructed, and the pathogenic virus infection is prevented, the pathogenic microorganisms are prevented more directionally and more actively, and the occurrence of the associated lentivirus of the EB virus is fundamentally prevented.

The research aims to research and screen EB virus antigen polypeptide capable of being combined with MHC class I and MHC class II molecules, and the EB virus antigen polypeptide is used for preparing an EB virus vaccine based on DC technology.

1. Prediction of the binding ability of EBV antigens to MHC class I and MHC class II molecules polypeptides:

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

2. From the above polypeptides, 50 polypeptide fragments with high predictive score and less predictive toxicity were selected and further studied. The specific experiment is as follows:

(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 killer activity assay was performed using T lymphocytes activated by induction of polypeptide-DCs as effector cells and EBV-expressing positive fibroblasts as target cells.

The method is divided into four groups: the treatment group polypeptide-DCs induce activated T cells, the control group DCs induce activated T cells according to the number of effector cells and target cellsThe quantity ratio is 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

Sequence of	Serial number
		CVVFPPTPV	SEQ ID NO:1
FPQCLPAYF	SEQ ID NO:2
		NATLIELCFR	SEQ ID NO:3
RSVSCFAVGY	SEQ ID NO:4
		TLIELCFRSV	SEQ ID NO:5
CFRSVSCFA	SEQ ID NO:6
		TLIELCFRSV	SEQ ID NO:7
AYFPSFHVL	SEQ ID NO:8
		SVSCFAVGYFFPQCL	SEQ ID NO:9
VSCFAVGYFFPQCLP	SEQ ID NO:10
		RSVSCFAVGYFFPQC	SEQ ID NO:11
FPPTPVLAVANATLI	SEQ ID NO:12
		FRSVSCFAVGYFFPQ	SEQ ID NO:13
SCFAVGYFFPQCLPA	SEQ ID NO:14
		TPVLAVANATLIELC	SEQ ID NO:15
PTPVLAVANATLIEL	SEQ ID NO:16

(3) Induced activation of T lymphocytes

Randomly drawing one of the 16 polypeptides, adding the obtained polypeptide-DCs into mitogen for incubation to enable the obtained polypeptide-DCs to lose the proliferation activity, washing the polypeptide-DCs for 2 times by PBS, mixing the polypeptide-DCs into a 96-hole culture plate according to the concentration ratio of DC to T of 1:20 for co-culture, dividing the polypeptide-DCs into four groups, namely a negative control group, a polypeptide group, a DC group and a polypeptide-DC group, changing the liquid every 2 days, adding the same amount of polypeptide-DCs when culturing the liquid is carried out till the fourth day, inducing the activation proliferation of specific CTL clone by repeated stimulation, and detecting the cell activity once by CCK-8 every two days from the first day after plating. Detection was by day 7. As shown in FIG. 2, the polypeptide-DC group was found to be most active.

3. Immunogenicity of EB Virus-specific DC vaccines

Immunogenicity was tested by randomly drawing one of the 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-EBV 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% positive, and the serum of the polypeptide vaccine group mouse is 60% 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 and induce an organism to generate the specific cellular immune response and the humoral immune response of the EBV antigen, thereby being expected to achieve the curative effect of preventing and treating the EBV-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

<110> Wittaen (Guangzhou) pharmaceutical Co., Ltd

<120> EB virus associated antigen short peptide and application thereof

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Claims

EB virus associated antigen short peptide, which is characterized in that the sequence is shown in any one of SEQ ID NO 1-16.
2. The use of the short peptide of claim 1 for the preparation of a DC vaccine for the control of epstein-barr virus-related lentivirus.
3. The use of the oligopeptide of claim 1 in the preparation of a medicament for the prevention and treatment of epstein-barr virus-related chronic diseases.
4. An epstein-barr virus antigen polypeptide DC vaccine loaded with the short peptides and dendritic cells of 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 one 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 a DC vaccine according to any one of claims 4 to 8 in the manufacture of a medicament for the prevention or treatment of chronic diseases associated with epstein-barr virus.