WO2021209035A1 - Novel coronavirus t cell epitope peptide, and pmhc and preparation and application thereof - Google Patents

Abstract

A novel coronavirus T cell epitope peptide, and pMHC and the preparation and application thereof. The amino acid sequence of the novel coronavirus T cell epitope peptide is as shown in any one of SEQ ID NOs: 1-9. Nine novel coronavirus T cell epitope peptides are discovered, and a pMHC compound monomer is prepared by using same; a pMHC compound polymer is further prepared, can be used for detecting antigen-specific T cells in peripheral blood of a novel coronavirus infected rehabilitative person, and is used for an in-vitro T cell activation experiment; the novel coronavirus T cell epitope peptides can be applied to immunodetection related to novel coronaviruses.

Description

A novel coronavirus T cell antigen epitope peptide, pMHC, and preparation and application thereof Technical field

The present invention relates to the field of biotechnology, and more specifically, to a novel coronavirus T cell epitope peptide, pMHC, and preparation and application thereof.

Background technique

The new coronavirus is the pathogen of the new infectious pneumonia that occurred at the end of 2019. It has caused a pandemic in more than 160 countries and regions around the world, with more than 15,000 deaths. At present, the source of the virus is not clear. Gene sequencing shows that it is highly homologous with bat SARS-like coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV). There are no reports of specific drugs. The immune response mechanism of the human immune system to the new coronavirus, including antigen recognition, immune clarification mechanism, and immune memory protection mechanism, are unclear.

According to the study of SARS and other coronaviruses with high homology of the new coronavirus, the T cell immune response plays an important role in the body's anti-viral defense and the body's immune pathological damage after virus infection, especially CD8 ⁺ T cells Its antigen-specific immune activity still exists after 11 years, which illustrates the ^{important role of CD8 +} T cell immune response in anti-coronavirus immune defense and its important position in vaccine development. The first step of CD8 ⁺ T cell immune response is that T cells specifically recognize the epitope presented by the virus-infected cell through the antigen recognition receptor on its surface. Therefore, the epitope is an important key molecule for T cells to specifically recognize the virus and exert immune protection, and it is a key target molecule for immune detection, immunotherapy and vaccine development.

The virus antigens in virus-infected cells need to be processed and presented by the antigen presentation system. After the virus antigen is degraded by the proteasome, it is combined with the transporter associated with antigen processing (TAP), and the antigen peptide of 8-12 amino acids is selectively transported by the TAP to the endoplasmic reticulum. The assembled main histocompatibility complex (MHC)-class I molecule's antigen binding groove is combined to form an antigen peptide-MHC-class I molecular complex (pMHC), and the complex is transported to the cell membrane through the Golgi apparatus. CD8 ⁺ T cells recognize pMHC activation through the T cell receptor (TCR), kill virus-infected cells and clear the virus, thereby exerting anti-virus cellular immunity. Therefore, computer simulation technology can be used to predict MHC-I (HLA-A2) epitopes from the parameters of the three key nodes of the proteasome, TAP and MHC, and to find T cells from the huge viral genome encoded proteins. Epitope.

There is no report or suggestion about T cell epitope and its application in immunoassay.

Summary of the invention

The purpose of the present invention is to provide a novel coronavirus T cell epitope peptide, pMHC, and preparation and application thereof.

The first objective of the present invention is to provide a novel coronavirus T cell epitope peptide

The second objective of the present invention is to provide a pMHC complex monomer containing the novel coronavirus T cell epitope peptide.

The third objective of the present invention is to provide a pMHC complex multimer containing the novel coronavirus T cell epitope peptide.

The fourth object of the present invention is to provide a method for preparing the pMHC complex monomer containing the novel coronavirus T cell epitope peptide.

The fifth object of the present invention is to provide the pMHC complex monomer prepared by the method.

The sixth object of the present invention is to provide a method for preparing a pMHC complex multimer containing the novel coronavirus T cell epitope peptide.

The seventh objective of the present invention is to provide the pMHC complex multimer prepared by the method.

The eighth object of the present invention is to provide said novel coronavirus T cell epitope peptide, said pMHC complex monomer, said pMHC complex multimer, and said pMHC complex The application of any one or more of the monomers and the pMHC complex multimers in the preparation of detection reagents for detecting new coronavirus.

The above-mentioned objects of the present invention are achieved through the following schemes:

T2-A2 is an antigen-presenting cell line expressing human MHC-I molecule HLA-A2 through recombinant genetic engineering technology. Only effective epitopes can be presented by it, thereby forming a stable pMHC complex on the cell surface, so it is one of the criteria for identifying and screening T cell epitopes.

T cell epitopes cannot be used alone, and must be developed in the form of pMHC monomers or multimers. The present invention utilizes genetically engineered recombinant HLA-A2 heavy chain and light chain proteins and the identified novel coronavirus T cell epitope to refold jointly to prepare pMHC monomers, and on this basis, prepare fluorescent markers The tetramer is found to be able to effectively recognize antigen-specific T cells in patients who have recovered from the new coronavirus infection, and effectively activate T cells. It proves that the newly discovered novel coronavirus T cell epitope has complete biological activity and can be used in immunoassays.

A novel coronavirus T cell antigen epitope peptide, whose amino acid sequence is shown in any one of SEQ ID NOs: 1-9.

SEQ ID NO:1: FVFLVLLPLV;

SEQ ID NO: 2: FQFCNDPFL;

SEQ ID NO: 3: YQDVNCTEV;

SEQ ID NO: 4: FTISVTTEI;

SEQ ID NO: 5: VVFLHVTYV;

SEQ ID NO: 6: YIWLGFIAGL;

SEQ ID NO: 7: RLNEVAKNL;

SEQ ID NO: 8: VMYSEFPAI;

SEQ ID NO: 9: GMALSHYYV.

The pMHC complex monomer of the novel coronavirus T cell epitope peptide.

The pMHC complex multimer of the novel coronavirus T cell antigen epitope peptide.

Preferably, the pMHC complex multimer is a tetramer.

More preferably, the tetramer is a fluorescently labeled tetramer.

The preparation method of the pMHC complex monomer of the novel coronavirus T cell antigen epitope peptide is to combine the HLA-A2 heavy chain protein, HLA-A2 light chain β2m protein and the novel coronavirus T cell antigen table Peptides are mixed and renatured and purified.

The renaturation is the reassembly of independent single-chain proteins into active protein complexes under specific conditions. This refers to the refolding of the three independent components of the MHC heavy chain, the MHC light chain and the antigen peptide into a pMHC complex. Here p stands for peptide, and MHC stands for histocompatibility complex.

Preferably, the molar ratio of HLA-A2 heavy chain protein, HLA-A2 light chain β2m protein and said novel coronavirus T cell epitope peptide is 1: (1-5): (1-20).

More preferably, the molar ratio of HLA-A2 heavy chain protein, HLA-A2 light chain β2m protein and said novel coronavirus T cell epitope peptide is 1:2:10.

Preferably, the purification is ion exchange column and molecular sieve purification.

The pMHC complex monomer prepared by the method.

A method for preparing a pMHC complex multimer containing the novel coronavirus T cell antigen epitope peptide, avidin, biotin, and the pMHC complex monomer are mixed and purified.

Preferably, the molar ratio of avidin, biotin and the pMHC complex monomer is 1: (1-8): (1-8).

More preferably, the molar ratio of avidin, biotin and the pMHC complex monomer is 1:4:4.

Preferably, the purification is molecular sieve purification.

The pMHC complex multimer prepared by the method.

The novel coronavirus T cell epitope peptide, the pMHC complex monomer, the pMHC complex multimer, the pMHC complex monomer, the pMHC complex The application of any one or more of the multimers in the preparation of new coronavirus detection reagents.

Epitopes can be assembled with HLA-A2 heavy chain and HLA-A2 light chain β2m protein into pMHC monomers, and further assembled into tetramers, and labeled with corresponding fluorescence. It can be used to detect T lymphocytes in the peripheral venous blood of the subject that can recognize the neocoronavirus antigen. It can be applied to:

1) Detect whether you are infected with the new coronavirus. Only after infection, the body produces an immune response before it produces antigen-specific T cells. Therefore, the detection of this T cell means that it has been infected with the new coronavirus.

2) Detect whether it has the cellular immune function to resist the new coronavirus infection. As above, the detection of neocoronavirus antigen-specific T cells in the examinee means that the body has developed T cell immune function. According to the ratio, the body can evaluate the body's immune function against neocoronavirus-infected T cells.

3) Monitor the condition. It can be used to monitor changes in the condition of close contacts, medical observers, suspected and confirmed patients.

4) Judgment of prognosis. If the body is unable to produce a T cell immune response, or the proportion of antigen-specific T cells continues to decrease, it indicates a poor prognosis.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention discovers 9 novel coronavirus T cell epitope peptides, and uses them to prepare pMHC complex monomers, and further prepares pMHC complex multimers, which can be used in the periphery of patients who have recovered from new coronavirus infections. The detection of antigen-specific T cells in the blood is used in in vitro T cell activation experiments. These novel coronavirus T cell epitope peptides can be applied to immunoassays related to the novel coronavirus.

Description of the drawings

Figure 1 shows the T2-A2 identification of nine epitopes on T cells of the novel coronavirus. A: Positive and negative control gradient experiment. Negative (black) is a negative irrelevant control, HLA-A2 does not bind; positive is other known positive polypeptides. B: T2-A2 identification experiment of new coronavirus antigen polypeptide. Sample 1: n-Sp1,2: n-Sp2, 6: n-Sp6, 7: n-Sp7, 9: n-Sp9, 11: n-Sp11, 13: n-Sp13, 16: o-Sp2, 18 : O-Sp4. C: Summary of three experimental repetitions.

Figure 2 shows the preparation of the pMHC complex of the novel coronavirus T cell epitope. A: Expression and affinity purification of HLA-A2 light chain recombinant protein. M: molecular marker; 1: light chain induced expression product; 2: chitin affinity chromatography washed light chain product; 3: chitin resuspended light chain; 4: DTT washed light chain; 5: light chain final product Eluted. B: Expression and affinity purification of HLA-A2 heavy chain recombinant protein. 6: heavy chain induced expression product; 7: chitin affinity chromatography to wash the heavy chain product; 8: chitin resuspends the heavy chain; 9: DTT washes the heavy chain; 10: heavy chain final product elution. C: Purification by DEAE cellulose ion exchange column (0.5M NaCl) after the pMHC complex is assembled. D: Molecular sieve (GE Superdex75pg) purification after assembly of the pMHC complex. E: Purification of molecular sieve (GE Superdex75pg) assembled by tetramer.

Figure 3 shows the immunodetection effect of the pMHC complex of the novel coronavirus T cell epitope n-Sp1. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared n-Sp1tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Figure 4 shows the immunodetection effect of the pMHC complex of the new coronavirus T cell epitope n-Sp2. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared n-Sp2tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Figure 5 shows the immunodetection effect of the pMHC complex of the n-Sp6 epitope of the novel coronavirus T cell antigen. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared n-Sp6tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Figure 6 shows the immunodetection effect of the pMHC complex of the n-Sp7 epitope of the novel coronavirus T cell antigen. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared n-Sp7tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Figure 7 shows the immunodetection effect of the pMHC complex of the novel coronavirus T cell epitope n-Sp9. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared n-Sp9tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Figure 8 shows the immunodetection effect of the pMHC complex of the new coronavirus T cell epitope n-Sp11. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared n-Sp11tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Figure 9 shows the immunodetection effect of the pMHC complex of the new coronavirus T cell epitope n-Sp13. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared n-Sp13tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Figure 10 shows the immunodetection effect of the pMHC complex of the novel coronavirus T cell epitope o-Sp2. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared o-Sp2tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Figure 11 Immunodetection effects of pMHC complexes of the o-Sp4 epitope of 11 novel coronavirus T cell antigens. The above-prepared pMHC complex tetramers were used for the flow cytometric detection of PBMC in the peripheral blood of HLA-A2-positive patients who had recovered from the novel coronavirus. A: Flow cytometry detection of pMHC complex tetramer (tetramer-PE) for detection of memory T cells in peripheral blood of patients who have recovered from new coronavirus infection. B: A summary of the results shows that the prepared o-Sp4tetramer can detect memory T cells in patients who have recovered from the novel coronavirus infection.

Detailed ways

The present invention will be further elaborated below in conjunction with specific embodiments, which are only used to explain the present invention and not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.

Example 1 Prediction of New Coronavirus HLA-A2 Restricted Epitope

1. Experimental method

Take the spike(S) and membrane of 2019-nCoV (MN908947.3), SARS Chinese isolate (DQ182595.1), MERS Riyadh isolate (KF600612.1) and 1997 American common coronavirus isolate (KF530099.1) (M) and nucleocapsid (N) protein sequences, compared with Clustal Omega. Select epitopes that are inconsistent with SARS, MERS and common coronavirus sequences for follow-up research. At the same time, a known HLA-A2 restricted CD8 ⁺ T cell epitope was selected as a positive control.

2. Experimental results

Screen a large number of high-efficiency and specific T cell epitope candidates.

Example 2 Identification of the new coronavirus HLA-A2 restricted epitope

1. Experimental method

The polypeptide predicted in Example 1 was artificially synthesized and configured into different concentrations, respectively 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. Take T2-A2 cells in logarithmic growth state, seeded into 96-well plates, ¹⁰⁵ per well, the distribution of holes arranged blank, negative control peptide (GLQRLGYVL, from Zika virus gene coding), a positive control peptide (influenza M1 polypeptide , GILGFVFTL) and each synthetic antigen polypeptide, each group has 3 multiple wells, the final volume is 200μL. Incubate at 37°C for 4 hours, centrifuge and wash twice, label with FITC anti-human HLA-A2 (β2m) antibody, incubate for 30 minutes in the dark at 4 degrees, and detect with flow cytometry. The experiment was carried out 3 times in total.

2. Experimental results

The results are shown in Figure 1. The results show that all 9 antigenic polypeptides can be effectively presented by antigen-presenting cells. The information is shown in Table 1.

Table 1: Novel coronavirus T cell epitopes

serial number	Code	length	sequence
1	n-Sp1	10	FVFLVLLPLV(SEQ ID NO:1)
2	n-Sp2	9	FQFCNDPFL(SEQ ID NO: 2)
6	n-Sp6	9	YQDVNCTEV(SEQ ID NO: 3)
7	n-Sp7	9	FTISVTTEI(SEQ ID NO: 4)
9	n-Sp9	9	VVFLHVTYV(SEQ ID NO: 5)
11	n-Sp11	10	YIWLGFIAGL(SEQ ID NO: 6)
13	n-Sp13	9	RLNEVAKNL (SEQ ID NO: 7)
16	o-Sp2	9	VMYSEFPAI(SEQ ID NO: 8)
18	o-Sp4	9	GMALSHYYV(SEQ ID NO: 9)

Example 3 Preparation of pMHC complex of neocoronavirus epitope

1. Preparation of HLA-A2 heavy chain and light chain

1. Experimental method

Recombinant construction of genetic engineering technology through SpeI and NdeI to link the heavy chain HLA-A2 (NCBI No. U02935.2) and light chain β2m (NCBI No. AY187687.1) into the prokaryotic expression vector pTXB1, respectively, to obtain pTXB1-HLA-A2h and pTXB1-β2m prokaryotic expression plasmids. The plasmid was transferred into BL21 Escherichia coli by heat shock method (stimulated at 42°C for 90 seconds) and sequenced and analyzed. The bacterial liquid identified by sequencing is stored at 30% glycerol at -20°C. Inoculate 10 μL of glycerol bacteria in 4 mL of LB medium containing ampicillin, culture at 37°C at 280 rpm for 16 to 18 hours, then expand the culture to 400 ml 37°C at 280 rpm for 4 hours, and induce expression by 0.1mM IPTG 24°C at 280 rpm for 4 hours. Ultrasonic break for 50 minutes (300W work for 3 seconds and stop for 5 seconds), centrifuge at 3000*g at 4°C for 15 minutes and collect the supernatant.

The centrifugal product filtered through a 0.45μm pore filter membrane passes through a chitin affinity chromatography column at low speed, and the chromatography is cleaned with 15 column volumes of loading buffer (20mM Tris-Hcl, 0.5M NaCl, pH8.5) Then quickly rinse the column with 2 times the column volume of elution buffer (50mM DTT loading buffer), and put the column in a refrigerator at 4℃. After 36 hours, the elution buffer is eluted and Collect the protein according to the OD ₂₈₀ nm ultraviolet absorption peak.

2. Experimental results

After purification, HLA-A2 heavy chain and light chain proteins were obtained (Figure 2A, B).

2. Preparation of pMHC complex monomer of epitope peptide

1. Experimental method

The HLA-A2 heavy chain, HLA-A2 light chain β2m and each epitope polypeptide in Example 2 were gradually added dropwise to the refolding solution (5M urea, 0.4M arginine) at a molar ratio of 1:2:10. Acid, 100mM Tris, 3.7mM cystamine, 6.3mM cysteamine, 2mM EDTA) were renatured to obtain the pMHC complex monomer of the epitope peptide. The pMHC complex monomer is further purified by a DEAE ion exchange column, eluted with 0.5M NaCl, and the protein is collected based on the OD280nm ultraviolet absorption peak. Then the protein purified by DEAE ion exchange column was purified by Superdex 75pg molecular sieve according to the molecular weight, eluted with PBS, and collected different molecular weight proteins according to the OD280nm ultraviolet absorption peak.

2. Experimental results

After purification, the pMHC complex monomer of the epitope peptide was obtained (Figure 2C, D).

3. Preparation of tetramer of fluorescently labeled neocoronavirus T cell antigen epitope (tetramer)

1. Experimental method

Since the expressed HLA-A2 has a biotin binding site, through the biotin and avidin system, the fluorescently labeled avidin (PE-streptavidin), biotin and pMHC complex monomers are in accordance with 1:4:4 The molar ratio is mixed at 4°C and incubated in the dark for 16-18 hours to prepare fluorescently labeled neocoronavirus T cell epitope tetramer. Purified by Superdex 75pg molecular sieve, eluted with PBS, and collected different molecular weight proteins according to the OD280nm ultraviolet absorption peak.

2. Experimental results

After purification, a fluorescently labeled neocoronavirus T cell epitope tetramer was obtained (Figure 2E).

Example 4 Application of Antigenic Epitopes in Patients Recovered from Novel Coronavirus Infection

1. Experimental method

Peripheral venous blood was collected from patients who recovered from the novel coronavirus infection during the reexamination, and peripheral blood mononuclear cells (PBMC) were isolated, and their HLA subtypes were identified. A total of 5 HLA-A2-positive recovered PBMC samples were obtained. The above-mentioned neocoronavirus T cell epitope polypeptide and the negative control polypeptide (GLQRLGYVL, coded from Zika virus gene) were selected separately by constructing their corresponding tetramers for detection.

2. Experimental results

The results showed that the 9 selected epitope tetramers can all recognize antigen-specific T cells produced in patients who have recovered from the novel coronavirus infection (Figure 3-Figure 11), and can be further developed as immunoassay reagents.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. For those of ordinary skill in the art, they can also make decisions based on the above descriptions and ideas. For other changes or changes in different forms, it is not necessary and impossible to enumerate all the implementation methods here. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims (9)

1. A novel coronavirus T cell antigen epitope peptide, characterized in that its amino acid sequence is shown in any one of SEQ ID NOs: 1-9.
2. A pMHC complex monomer containing the novel coronavirus T cell epitope peptide of claim 1.
3. A pMHC complex polymer containing the novel coronavirus T cell epitope peptide of claim 1.
4. The pMHC complex multimer of claim 3, which is a tetramer.
5. A method for preparing a pMHC complex monomer containing the novel coronavirus T cell epitope peptide according to claim 1, characterized in that the HLA-A2 heavy chain protein, HLA-A2 light chain β2m protein and the claim The said novel coronavirus T cell epitope peptides described in 1 are mixed and renatured and purified.
6. The pMHC complex monomer prepared by the method of claim 5.
7. A method for preparing a pMHC complex multimer containing the novel coronavirus T cell epitope peptide of claim 1, characterized in that avidin, biotin, and the pMHC complex of claim 2 are single The aggregates are mixed and purified.
8. The pMHC complex multimer prepared by the method of claim 7.
9. The novel coronavirus T cell epitope peptide according to claim 1, the pMHC complex monomer according to claim 2, the pMHC complex multimer according to claim 3, and the pMHC complex multimer according to claim 6 The use of any one or more of the pMHC complex monomers and the pMHC complex multimers of claim 8 in the preparation of a new coronavirus detection reagent.

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