CN117384262A - Mycobacterium tuberculosis specific T cell HLA-A 0201 restriction epitope peptide pMtb8 and application thereof - Google Patents
Mycobacterium tuberculosis specific T cell HLA-A 0201 restriction epitope peptide pMtb8 and application thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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Abstract
The invention discloses a mycobacterium tuberculosis specific T cell HLA-A x 0201 restriction epitope peptide pMtb8 and application thereof, belonging to the field of biotechnology and medicine. The invention combines a bioinformatics prediction method and an ultraviolet induced peptide replacement experiment, utilizes the antigen of mycobacterium tuberculosis to screen out epitope peptide with antitubercular activity, and efficiently and accurately detects the affinity between epitope peptide pMtb8 and HLA-A 0201 molecules. This is a novel, unreported HLA-A 0201-restricted mycobacterium tuberculosis epitope peptide, specifically CD8 in tuberculosis patients + T cells recognize and induce activation. HLA-A x 0201 genotype is one of the most widely carried HLA genotypes in humans, the inventionThe determination of epitope peptide provides theoretical basis and new solution for research and development of tuberculosis vaccine, diagnostic reagent and therapeutic drug, and has practical significance for preventing and treating tuberculosis.
Description
Technical Field
The invention belongs to the field of biotechnology and medicine, and in particular relates to mycobacterium tuberculosis specific CD8 + T cell HLA-A 0201 restriction epitope peptide pMtb8And its application.
Background
Tuberculosis is an infectious disease caused by infection with mycobacterium tuberculosis (Mycobacterium tuberculosis, mtb) and having a very high global infection rate. The main site of infection of Mtb is the lung, causing tuberculosis. In addition, mtb can also infect other organs and tissues, such as lymph nodes, brain, kidneys, spine, etc., with extrapulmonary tuberculosis. According to world health organization (World Health Organization, WHO) data, about 1/4 of the global population had or was contracting Mtb, and only 2020, about 1000 tens of thousands of cases of active tuberculosis were developed. Nowadays, tuberculosis epidemic situation presents new characteristics, namely more and more drug-resistant tuberculosis cases and more tuberculosis and AIDS co-infection conditions. There is currently no effective prophylactic measure for both types of patients. China is one of the most heavily loaded countries in the world, and research and development of tuberculosis vaccines and therapeutic drugs suitable for Chinese people have important significance for public health care and protection of people's health.
Mtb is an intracellular parasitic bacterium for which effective control by humans is primarily dependent on cellular immunity, particularly T cell immunity. T cells in humans are largely divided into CD4 + T (Th) cells and CD8 + Numerous studies have demonstrated that two large subpopulations of T (CTL) cells, both of which have important roles in controlling Mtb infection, CD8 + T is the principal force for eliminating tubercle bacillus. T cells are activated by recognizing epitope peptides presented by antigen presenting cells, eliciting an anti-tuberculosis immune response. Epitope peptides are the core of the protein antigen sequence that elicit an immune response, also known as antigenic determinants, that bind to MHC (HLA) molecules on the surface of antigen presenting cells, form peptide-HLA complexes, and are delivered and expressed on the surface of antigen presenting cells (e.g., dendritic cells, B lymphocytes, etc.), thereby activating T cells. HLAI class molecules present epitope peptides to CD8 + T cells, HLAII class molecules present epitope peptides to CD4 + T cells. Epitope peptides form stable complexes with HLA molecules to effectively activate T cells, and affinity of peptides to HLA molecules is critical for the formation of peptide-HLA complexes. Thus, T cell epitope peptides with immune effect are obtained, the first step being to screen out high affinityAnd further identifying an epitope peptide having immunological activity.
At present, only one vaccine for preventing tuberculosis is used clinically, and the vaccine has better preventing effect on children tuberculosis, especially severe tuberculosis such as tuberculous meningitis, but has poorer preventing effect on adult tuberculosis. At present, research on novel vaccines at home and abroad is hot, and novel vaccines based on epitope peptides are one of hot spots. The epitope peptide can be completely obtained through artificial synthesis, and the research and development period is short; the safety is good because other harmful components of pathogens except the active sequence are removed; multiple epitope peptides can also be used in tandem to enhance immune effects and reduce immune escape. In addition, the epitope peptide can be used for preparing MHC tetramers, and is applied to multiple aspects such as immunological research and detection, specific immunotherapy, vaccine curative effect monitoring and the like. HLA-A 0201 is one of the most common HLA molecules, and the identification of HLA-A 0201 restriction epitope has wide application population, so that the method has very important significance.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a mycobacterium tuberculosis specific CD8 + T cell HLA-A x 0201 restriction epitope peptide pMtb 8.
Another object of the present invention is to provide the Mycobacterium tuberculosis specific CD8 + Application of T cell HLA-A 0201 restriction epitope peptide pMtb8 in preparing tuberculosis vaccine, diagnostic reagent and therapeutic medicine.
The aim of the invention is achieved by the following technical scheme:
the invention provides a polypeptide which has high affinity with HLA-A x 0201 molecules and can activate CD8 + T cells, mycobacterium tuberculosis specific CD8 inducing proliferation and secretion of IFN-gamma + A T cell epitope peptide designated pMtb8, the amino acid sequence of said epitope peptide being:
pMtb 8: GLAEVFAV (SEQ ID NO. 8) (i.e., gly-Leu-Ala-Glu-Val-Phe-Ala-Val);
the invention also provides the mycobacterium tuberculosis specific CD8 + T cell epitope peptide pMtb8 in preparation of tuberculosis diagnostic reagentIs used in the application of (a).
The invention also provides the mycobacterium tuberculosis specific CD8 + Application of T cell epitope peptide pMtb8 in preparing medicament for treating tuberculosis.
The invention also provides the mycobacterium tuberculosis specific CD8 + Use of T cell epitope peptide pMtb8 for the preparation of a prophylactic/therapeutic vaccine for tuberculosis.
The medicine is stimulated HLA-A 0201 tuberculosis patient CD8 + T cell proliferation drugs.
The medicine is a medicine for stimulating IFN-gamma secretion cell proliferation.
The medicine is a medicine for inducing a cellular immune response against tuberculosis.
A tuberculosis diagnosis kit comprises the mycobacterium tuberculosis specific CD8 + T cell epitope peptide pMtb 8.
A pharmaceutical composition comprises the above mycobacterium tuberculosis specific CD8 + T cell epitope peptide pMtb 8.
Preferably, the medicaments which are the same or different can be prepared into various medicinal dosage forms by adopting a conventional method, and the dosage forms comprise: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, medicinal granules, pills, pellets, suspensions, wines, tinctures, drops and other oral administration forms, and injection and other oral administration forms, such as injection and the like.
Preferably, the medicaments, which are the same or different, respectively, can also contain one or more pharmaceutically acceptable carriers or excipients.
Preferably, the excipient may include diluents, wetting agents, lubricants, fillers, preservatives, and the like.
Compared with the prior art, the invention has the following advantages and effects:
(1) Vaccines remain the best strategy for controlling tuberculosis epidemics. The BCG vaccine used clinically at present is only effective in preventing severe tuberculosis of children, and has no direct protection effect on adults. Thus, there is an urgent need to develop new vaccines-! Polypeptide vaccines developed based on epitopes have several important advantages: firstly, the polypeptide vaccine has a unique advantage, namely, an epitope with immune advantage can be selected, and adverse reactions such as autoimmunity and the like caused by cross reaction of thalli or protein vaccine and self tissues are avoided; second, synthetic peptides do not contain genetic material nor do they have the problem of restoring virulence, both of which are worrying about attenuated vaccines and mutants; thirdly, the polypeptide epitope vaccine can induce more immune response than the protein vaccine, and can avoid concentrated immunity induced by the protein vaccine; in addition, the epitope peptide can be completely obtained through artificial synthesis, the research and development period is short, and a plurality of epitope peptides can be used in series, so that the immune effect is enhanced and the immune escape is reduced. However, the epitope conformation is complex, accurate screening has limitation, immunogenicity is low, and adjuvant assistance is needed to generate a good immune response. The currently discovered tuberculosis CTL epitope peptide is mainly identified in European and American population, but the effect of the currently discovered tuberculosis CTL epitope peptide on Asian population with severe tuberculosis epidemic is unknown. The invention has important significance for research and development of anti-tuberculosis polypeptide vaccine and diagnostic reagent.
(2) The invention combines a bioinformatics prediction method and an ultraviolet induced peptide replacement experiment, utilizes the antigen of mycobacterium tuberculosis to screen out epitope peptide with antitubercular activity, and efficiently and accurately detects the affinity between epitope peptide pMtb8 and HLA-A 0201 molecules. This is a novel, unreported HLA-A 0201-restricted mycobacterium tuberculosis epitope peptide, specifically CD8 in tuberculosis patients + T cells recognize and induce activation. HLA-A 0201 genotype is one of the most widely carried HLA genotypes in the population, and the determination of the epitope peptide provides theoretical basis and new solution for the research and development of tuberculosis vaccines, diagnostic reagents and therapeutic drugs, and has practical significance for preventing and treating tuberculosis.
Drawings
FIG. 1 is a schematic representation of the affinity of the candidate epitope peptide pMtb8 for HLA-A x 0201 molecules.
FIG. 2 is a mass spectrometry diagram of the sequence of the epitope peptide pMtb 8.
FIG. 3 is a schematic representation of ELISPOT detection of IFN-gamma secretion by T cells of a patient suffering from pMtb 8-induced tuberculosis.
FIG. 4 shows the detection of CD8 in patients suffering from pMtb 8-induced tuberculosis in CFSE-based marker proliferation assay + Results of T cell proliferation are schematically shown.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.
The invention comprehensively utilizes various technical means of bioinformatics and immunoinformatics to limit CD8 of HLA-A 0201 of mycobacterium tuberculosis antigen protein + The T epitope peptide is subjected to predictive analysis, the binding affinity and stability of the candidate peptide and HLA-A x 0201 molecules are checked by adopting an ultraviolet induced peptide replacement experiment, the epitope peptide is obtained by screening, and the functional effect of the epitope peptide is identified by an immunological experiment. Screening results found CD8 + T epitope peptide pMtb8 capable of inducing HLA-A x 0201 type tuberculosis patient CD8 + T cells produce a strong cellular immune response, secrete high levels of IFN-gamma, and induce CD8 + T cells proliferate significantly.
Example 1: acquisition of HLA-A x 0201 restriction epitope peptide
Using bioinformatics methods, HLA-A x 0201 restriction epitope peptides were predicted:
frequency distribution information for each genotype of the HLA-a locus of the population is obtained from HLA gene frequency database AFND (Allele Frequency Net Database, AFND), wherein HLA-a x 0201 is about 27% allele frequency in european population, about 11% carrier frequency in chinese population, and is one of the highest frequency HLA-a genotypes.
Through investigation of documents, mycobacterium tuberculosis antigen proteins with stronger immunogenicity are screened, 30 total specific protein names are shown in Table 1. The amino acid sequences of the respective antigen proteins were obtained from GeneBank.
TABLE 1 candidate Mycobacterium tuberculosis antigen proteins
Predicting HLA-A x 0201-restricted CD8 contained in antigenic proteins using bioinformatics algorithms + T epitope peptide, the algorithm adopted is NetMHCons in CBS database. Ranking the predictions according to IC50 values, lower IC50 values indicate higher affinity of the epitope peptide to HLA molecules, and lower IC50 values are generally considered to be indicative of high affinity. From this, HLA-A x 0201 restricted epitope peptides out of 30 antigen proteins were predicted and high affinity epitope peptides were screened out as candidate epitope peptides as shown in table 2.
TABLE 2 HLA-A 0201 restriction candidate epitope peptides
And detecting the affinity and the binding stability between the candidate epitope peptide and the HLA-A 0201 molecule by using an ultraviolet induced peptide substitution experimental method. The experimental procedure was as follows:
under ultraviolet irradiation, the peptide-HLA-0201 complex containing the photosensitive epitope peptide breaks, the broken peptide segment is not combined with HLA-0201 any more, and is separated from HLA-0201 molecules, at the moment, the candidate epitope peptide to be detected is added into a reaction system, if the candidate epitope peptide and the HLA-0201 molecules have higher affinity, new stable epitope peptide-HLA complex can be formed, otherwise, if the candidate epitope peptide and the HLA-0201 molecules do not have higher affinity, the new stable epitope peptide-HLA complex cannot be formed, and meanwhile, the HLA-0201 molecules losing the epitope peptide are unstable and rapidly degraded. The HLA molecules were captured using anti- β2m antibodies, and ELISA was used to detect the concentration of newly formed epitope peptide-HLA complexes and to evaluate the affinity between epitope peptide and HLA molecules. Positive peptide was used as positive control (pos.control), negative peptide was used as negative control (neg.control), and No epitope peptide was added as blank control (No peptide); the amino acid sequence of the positive peptide is NLVPMVATA; the amino acid sequence of the negative peptide was CTELKLSDY. Referring to positive peptides (pos.control), binding force greater than 100% indicates that the candidate peptide binds to HLA-A x 0201 more than positive peptide, i.e., high binding force peptide. Wherein, the binding force of pMtb8 and HLA-A 0201 is 211.4%, as shown in fig. 1 and table 3.
TABLE 3 high affinity binding of pMtb8 to HLA-A 0201
Experimental results:
the amino acid sequence of the epitope peptide pMtb8 is GLAEVFAV (SEQ ID NO. 8), and the mass spectrum analysis chart is shown in FIG. 2. Bioinformatics prediction result, binding force IC50 value of epitope peptide pMtb8 and HLA-A x 0201 is 3.66nM; the ultraviolet external exchange experiment detection shows that the epitope peptide pMtb8 and HLA-A 0201 can form a stable compound, and the binding force is 211.4%; it can be seen that the epitope peptide pMtb8 has a very high affinity with HLA-A x 0201 molecules.
Example 2: ELISPot experiment to detect secretion of IFN-gamma by T cells induced by epitope peptide pMtb8
The experimental procedure was as follows:
peripheral blood mononuclear cells (peripheral blood mononuclear, PBMCs) from patients with HLA-A x 0201 genotype tuberculosis were isolated using a density gradient method. Diluting 20mL of collected peripheral blood by one time with PBS, slowly adding the diluted peripheral blood into the upper part of lymphocyte separation liquid, wherein the volume ratio of the two is 2:1, centrifuging at 800g/min for 30min, separating the centrifuged liquid into three layers, wherein PBMC is arranged between the first layer and the second layer to form Bai Mozhuang, carefully sucking the PBMC into a new centrifuge tube by using a dropper, adding more than 5 times of volume of PBS for washing, centrifuging at 800g/min for 10min, repeatedly washing once, freezing cells by using frozen stock (90% FBS+10% DMSO), and storing by using liquid nitrogen.
ELISpot experiment: recovery of PBMCs from patient with HLA-A 0201 genotype at 37 ℃,5% CO 2 The incubator was allowed to stand overnight. Dead cells in PBMCs were removed with decellularized magnetic beads. Thin and fineCell count, complete medium resuspended cells to 1.25X10 6 mu.L/well of cell suspension was plated onto ELISPot plates (i.e., 2.5X10) 5 And/well) 48 wells. Epitope peptide pMtb8 was added to wells at a concentration of 10. Mu.g/mL, positive control wells were each added with 4. Mu.g/mLPHA (phytohemagglutinin), and 2 negative control wells were supplemented with whole culture without any stimulation. After each well was loaded with a sample, the plates were placed in a cell incubator at 37℃with 5% CO 2 Culturing for 24h. Taking out the ELISPot plate, washing for 5 times by using PBS, adding 100 mu L of enzyme-labeled anti-IFN-gamma antibody after beating, incubating for 2 hours, washing for 5 times by using PBS, adding 100 mu L of color-developing agent after beating, and stopping the reaction by using ultrapure water after obvious spots appear in positive control holes; airing, reading the plate, counting and correcting. Peptide specific T cell frequency at SFC/10 6 And (3) representing.
Experimental results: FIG. 3 shows secreted IFN-gamma antigen specific CD8 detected by ELISPOT following stimulation of HLA-A x 0201 tuberculosis patients by the epitope peptide pMtb8 + Frequency of T cells. As can be seen from the figure, in PBMC of HLA-A x 0201 tuberculosis patient, pMtb 8-specific CD8 + T cell frequencies were significantly higher than Negative Control (NC).
Example 3: CFSE-labeled lymphocyte proliferation assay to detect induction of CD8 by epitope peptide pMtb8 + T cell proliferation
The experimental procedure was as follows:
PBMCs of HLA-A 0201 type tuberculosis patients were resuscitated and allowed to stand overnight. Dead cells were removed using a dead cell-removing magnetic bead, counted, centrifuged and resuspended to 1×10 with PBS 7 mu.M CFSE label was added to each mL, and incubated for 10min in the dark after mixing. Staining markers were terminated by adding 5 volumes of pre-chilled complete medium (10% FBS+RPMI-1640). Centrifugation at 300g for 10min, washing with complete medium, and repeating twice. The cells were resuspended to 1X 10 with complete medium and counted again 6 And each mL. The cell suspension was plated into 96-well U-bottom cell culture plates at 200. Mu.L/well for a total of 8 wells, 10. Mu.g/mL of epitope peptide pMtb8 was added to each well of the experimental group, 4. Mu.g/mL PHA was added to the positive control well, and no stimulus was added to the negative control well, supplementing Quan Pei. Cells were identified as CFSE labeled (green fluorescence) by observation under a fluorescence microscope. 37 ℃,5% CO 2 Culturing in incubator for 48 hr. Each hole is divided into100IU of IL-2 is added respectively, the culture is continued until the 7d is reached, the cells are taken out, the anti-CD8 antibody is marked after washing, and the flow cytometry detection is carried out.
According to the characteristic of CFSE dye marking, the dye is uniformly distributed from the parent cells to the daughter cells along with cell proliferation, and the fluorescence intensity is halved. Therefore, cells with weak CFSE fluorescence are proliferated cells. Epitope peptide induced CD8 + CD8 whose T cell proliferation capacity is fluorescent by weak CFSE + T cell/CD 8 + T cell representation.
Experimental results: the detection results are shown in FIG. 4, from which it can be seen that epitope peptide pMtb8 can induce HLA-A x 0201 type tuberculosis patient CD8 + T cells proliferate specifically, whereas healthy volunteers do not proliferate significantly. This indicates that pMtb8 has a strong immunogenicity and is capable of inducing CD8 + T cell activation and expansion of CD8 + T cell response.
In summary, the epitope peptide pMtb8 identified by the screening of the present invention is specifically recognized by HLA-A x 0201 type tuberculosis patients and induces CD8 + The proliferation and secretion of IFN-gamma by T cells provide theoretical basis and new solution for the research and development of tuberculosis vaccines, diagnostic reagents and therapeutic drugs, and have great significance for the prevention and treatment of tuberculosis. If the epitope peptide pMtb8 of the invention can be directly used as a vaccine for tuberculosis, the epitope peptide pMtb8 can also be used as a component of a medicament for treating tuberculosis; it is also possible to determine whether a patient has tuberculosis by detecting the patient's immune response to the epitope peptide pMtb8, so that the epitope peptide pMtb8 can also be used as a component in a tuberculosis diagnostic kit.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. Mycobacterium tuberculosis specific CD8 + The T cell epitope peptide pMtb8 is characterized in that the amino acid sequence of the epitope peptide pMtb8 is shown in SEQ ID NO. 8.
2. Mycobacterium tuberculosis specific CD8 as described in claim 1 + Use of the T cell epitope peptide pMtb8, characterized by one of the following:
1) Mycobacterium tuberculosis specific CD8 as described in claim 1 + Application of T cell epitope peptide pMtb8 in preparing tuberculosis diagnostic reagent;
2) Mycobacterium tuberculosis specific CD8 as described in claim 1 + Application of T cell epitope peptide pMtb8 in preparing medicament for treating tuberculosis;
3) Mycobacterium tuberculosis specific CD8 as described in claim 1 + Use of T cell epitope peptide pMtb8 for the preparation of a prophylactic/therapeutic vaccine for tuberculosis.
3. The use according to claim 2, characterized in that: the medicine is stimulated HLA-A 0201 tuberculosis patient CD8 + T cell proliferation drugs.
4. The use according to claim 2, characterized in that: the medicine is a medicine for stimulating IFN-gamma secretion cell proliferation.
5. The use according to claim 2, characterized in that: the medicine is a medicine for inducing a cellular immune response against tuberculosis.
6. Use according to any one of claims 2-5, characterized in that: the medicament is in the form of tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquid, buccal agents, granules, medicinal granules, pills, pellets, suspension, wine, tincture, drops or injection.
7. Use according to any one of claims 2-5, characterized in that: the medicine also contains one or more pharmaceutically acceptable carriers or excipients.
8. The use according to claim 7, characterized in that: the excipient includes diluent, wetting agent, lubricant, filler or preservative.
9. A tuberculosis diagnostic kit, characterized in that: comprising the Mycobacterium tuberculosis specific CD8 of claim 1 + T cell epitope peptide pMtb 8.
10. A pharmaceutical composition characterized by: comprising the Mycobacterium tuberculosis specific CD8 of claim 1 + T cell epitope peptide pMtb 8.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20110236411A1 (en) * | 2007-09-27 | 2011-09-29 | Dako Denmark A/S | MHC Multimers in Tuberculosis Diagnostics, Vaccine and Therapeutics |
| CN104387450A (en) * | 2014-10-29 | 2015-03-04 | 南方医科大学 | Mycobacterium tuberculosis specific CD8<+>T cell epitope peptide P12 and application thereof |
| CN106248935A (en) * | 2016-08-31 | 2016-12-21 | 中国疾病预防控制中心传染病预防控制所 | Antigen of mycobacterium tuberculosis albumen Rv1798 and the application of t cell epitope peptide thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110236411A1 (en) * | 2007-09-27 | 2011-09-29 | Dako Denmark A/S | MHC Multimers in Tuberculosis Diagnostics, Vaccine and Therapeutics |
| CN104387450A (en) * | 2014-10-29 | 2015-03-04 | 南方医科大学 | Mycobacterium tuberculosis specific CD8<+>T cell epitope peptide P12 and application thereof |
| CN106248935A (en) * | 2016-08-31 | 2016-12-21 | 中国疾病预防控制中心传染病预防控制所 | Antigen of mycobacterium tuberculosis albumen Rv1798 and the application of t cell epitope peptide thereof |
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