CN110804088A - Cytomegalovirus-associated antigen short peptide and application thereof - Google Patents
Cytomegalovirus-associated antigen short peptide and application thereof Download PDFInfo
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
- C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention discloses a cytomegalovirus related antigen short peptide and application thereof. The sequence of the cytomegalovirus related antigen short peptide 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. The DC vaccine prepared by sensitizing at least one in-vitro dendritic cell in the short peptide can prevent cytomegalovirus related diseases, 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
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, it relates to cytomegalovirus-associated antigen short peptides and uses thereof.
Background
Human Cytomegalovirus (HCMV), HHV-5, is the most common and most harmful virus of intrauterine infections. HCMV has a typical herpes virus morphology with a DNA structure very similar to that of herpes simplex virus (herpesvirus), but 5% greater than HSV. The virus has high species specificity to a host, the infected host is narrow, and Human Cytomegalovirus (HCMV) only can infect human and can proliferate in human fiber cells. It has been shown that HCMV can cause the development of chronic diseases, and in patients with atherosclerosis, plaque detection can detect that the expression of HCMV DNA is increased; the existence of HCMV genome can be found by lymphocyte gene detection of diabetes patients; HCMV infection is more frequent in hypertensive patients, HCMV is also associated with hypertension progression, higher HCMV antibodies in vivo are higher in patients with higher blood pressure or with more target organ damage; the positive follow-up of the HCMV antibody finds that the incidence rate of the Alzheimer disease is higher, and the Alzheimer disease is possibly related to immune and inflammatory mechanisms; HCMV is also closely associated with a variety of tumors, such as breast, ovarian, colorectal, etc., and its positive expression correlates with malignancy and can affect tumor prognosis.
At present, no effective treatment method exists for HCMV, and the early prevention of protective vaccines becomes a research hotspot in recent years, so far, no effective vaccine exists.
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. Cytomegalovirus 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 cytomegalovirus vaccine, provide cytomegalovirus antigen polypeptide capable of being combined with MHC class I and MHC class II molecules, provide a HCMV vaccine based on DC technology, be applied to preventing and treating HCMV related chronic diseases by adopting the DC technology, increase the activity of mature DC cells by combining stem cell factor/vitrogen, promote the DC cell activation by a plurality of HCMV specific antigen peptides, achieve the purposes of preventing cytomegalovirus infection and treating, and have good application prospect.
The invention aims to provide HCMV antigen-associated short peptide.
The invention also aims to provide application of the HCMV antigen-related short peptide in preparing an HCMV antigen polypeptide DC vaccine.
The invention further aims to provide the HCMV antigen polypeptide DC vaccine.
The above purpose of the invention is realized by the following technical scheme:
provides HCMV related antigen short peptide, the sequence of which is shown in any one of SEQ ID NO 1-16.
Provides the application of the HCMV related antigen short peptide in the preparation of cytomegalovirus antigen polypeptide DC vaccine.
Provides the application of the HCMV-associated antigen short peptide in preparing the prevention and treatment medicine for HCMV-associated diseases.
In addition, the DC vaccine for preventing and treating the cytomegalovirus-related lentivirus is obtained by loading the cytomegalovirus-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 cytomegalovirus associated 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 cytomegalovirus-related slow diseases, the stem cell factor or the vitrogen factor is selected as an adjuvant to increase the activity of DC cells, and the specific antigen peptide sensitizes dendritic cells in vitro to obtain an autologous dendritic cell preparation which is used as a venous return type vaccine for preventing and treating the cytomegalovirus-related slow diseases. 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 cytomegalovirus-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 prevention and treatment medicine for the cytomegalovirus related chronic diseases also belongs to the protection scope of the invention.
The invention predicts that HCMV can be used as a sequence cluster of antigen by combining with bioinformatics technology, and after a large number of researches and searches, the obtained 16 cytomegalovirus 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 vaccine and DC vaccine, and prompt that the HCMV 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 cytomegalovirus and CTL cells aiming at the specificity of the cytomegalovirus, and thus can effectively prevent and treat the occurrence and development of cytomegalovirus related chronic diseases.
The invention has the following beneficial effects:
the invention provides cytomegalovirus 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 cytomegalovirus 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 cytomegalovirus based on the cytomegalovirus 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 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. HCMV 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 the body is reconstructed, and the pathogenic virus infection is prevented, the pathogenic microorganisms are prevented more directionally and actively, and the cytomegalovirus related lentivirus is fundamentally prevented.
The aim of the research is to develop and screen cytomegalovirus antigen polypeptides capable of combining with MHC class I and MHC class II molecules, and the cytomegalovirus antigen polypeptides are used for preparing a cytomegalovirus vaccine based on DC technology.
1. Prediction of the ability of HCMV antigens to bind to MHC class I and MHC class II molecules polypeptides:
through uniprot data base, determining HCMV sequence cluster (https:// www.uniprot.org/uniref /), inputting the sequence cluster (http:// www.ddg-pharmac. net/VaxiJen. html), analyzing the sequence cluster on line, setting Threshold to be 0.6, determining HCMV antigen protein, and predicting short peptide sites with high affinity with MHC class I and MHC class II molecules through IEDB data base 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 polypeptide-DCs-induced activated T lymphocytes as effector cells and HCMV-expressing positive fibroblasts 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
| Sequence of | Serial number |
| VPVPSFQPLI | SEQ ID NO:1 |
| RLTASSSAK | SEQ ID NO:2 |
| LTASSSAKR | SEQ ID NO:3 |
| TSSGTAAKR | SEQ ID NO:4 |
| RTSSGTAAK | SEQ ID NO:5 |
| VPVPSFQPL | SEQ ID NO:6 |
| RTSSGTAAK | SEQ ID NO:7 |
| KRAAAPLSSVSSNRL | SEQ ID NO:8 |
| RAAAPLSSVSSNRLT | SEQ ID NO:9 |
| MFHALAGRGRLLLLV | SEQ ID NO:10 |
| FHALAGRGRLLLLVP | SEQ ID NO:11 |
| AAAPLSSVSSNRLTA | SEQ ID NO:12 |
| QPLIRARGTKSGGDL | SEQ ID NO:13 |
| FQPLIRARGTKSGGD | SEQ ID NO:14 |
| PVPSFQPLIRARGTK | SEQ ID NO:15 |
| SVSSNRLTASSSAKR | 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 HCMV-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.
Serum anti-HCMV IgG antibody levels were detected by ELISA kit, results are shown in fig. 3, both DC vaccine group and blank group were negative; all the polypeptide-DC groups are positive, and 80% of the mouse serum of the polypeptide vaccine group turns 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 a role in antigen presentation and induce an organism to generate specific cellular immune response and humoral immune response of HCMV antigen, thereby being expected to achieve the curative effect of preventing and treating HCMV-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
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Claims (9)
1. The cytomegalovirus associated antigen short peptide is characterized in that the sequence of the cytomegalovirus associated antigen short peptide 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 prevention and treatment of cytomegalovirus-associated lenti-diseases.
3. The use of the short peptide of claim 1 in the preparation of a medicament for the prevention or treatment of chronic diseases associated with cytomegalovirus.
4. A cytomegalovirus antigen polypeptide DC vaccine loaded with the short peptide of claim 1 and dendritic cells.
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 the DC vaccine of any one of claims 4-8 in the manufacture of a medicament for the prevention and treatment of chronic diseases associated with cytomegalovirus.
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| CN115894708A (en) * | 2022-08-16 | 2023-04-04 | 青岛大学 | Human cytomegalovirus epitope chimeric peptide and application thereof |
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