CN111440229B

CN111440229B - Novel coronavirus T cell epitope and application thereof

Info

Publication number: CN111440229B
Application number: CN202010287172.7A
Authority: CN
Inventors: 张纪岩; 李伍举; 刘涛; 程倩倩; 张耀林; 侯春梅; 董洁; 王庆阳; 林周; 杨锡琴
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2020-04-13
Filing date: 2020-04-13
Publication date: 2021-08-03
Anticipated expiration: 2040-04-13
Also published as: CN111440229A

Abstract

The invention discloses a novel coronavirus T cell epitope and application thereof. The T cell epitope of SARS-CoV-2N protein is predicted by utilizing IEDB resources, and an immune mouse is verified, and the fragments 12-20, 49-57,104-112,127-135 have the T cell epitope effect, wherein the fragment 127-135 has the strongest effect. The research result of the invention has important significance for researching and developing SARS-CoV-2 vaccine and monitoring epidemic situation.

Description

Novel coronavirus T cell epitope and application thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to a novel coronavirus T cell epitope and application thereof.

Background

The novel coronavirus pneumonia has high spreading risk in the global range and poses great threat to human health safety. The novel coronavirus (SARS-CoV-2) is the 7 th coronavirus known to infect human, belongs to the genus coronavirus beta, has linear single-strand positive-strand RNA as genome, and is generally susceptible to human. The new coronary pneumonia has certain similarity with SARS and MERS infected by coronavirus of the same genus in pathological characteristics, and the 2019-nCoV has 70% and 40% gene sequence similarity with SARS-CoV and MERS-CoV respectively. Early cases suggest that 2019-nCoV infection may be less severe than symptoms caused by SARS-CoV and MERS-CoV infection. However, the number of attacks increases rapidly and there is increasing evidence of interpersonal transmission that the virus is more infectious than SARS-CoV and MERS-CoV.

The human immune system is a complex system composed of multiple organs, multiple immune cells and various immune molecules. They work together to build a layer-by-layer line of defense against various pathogens (viruses, bacteria, parasites, etc.). Innate immunity is the non-specific immune system and the adaptive immune system is required to produce a specific immune response to act more efficiently against a particular pathogen. Antibodies or vaccines, are closely related. B cells and T cells are the main "arms" in adaptive immunity. Unlike in innate immunity, the soldiers of these arms can recognize and destroy a particular pathogen. Meanwhile, some soldiers can remember the appearance of a target enemy, and once the same enemy invades again, the soldiers can quickly sound an alarm to initiate a wipe-out battle on the invader.

Mature B cells carry a detector called B cell receptor, which proliferate and differentiate upon detection of the corresponding antigen with the help of helper T cells. One part was differentiated into plasma cells capable of producing antibodies, and the other part became memory B cells. The antibody has the same detector as the B cell that produced it, patrols in body fluids, and labels those specific pathogens or blocks them from infecting human cells directly. It is this mechanism that is exploited by vaccines to elicit the production of antibodies by B cells to defend against foreign enemies through antigenic information from pathogens.

T cells are another important class of specific immune cells. The main function of helper T cells is to regulate or assist other immune cells in functioning by releasing cytokines after recognizing antigens, such as assisting in activating B cells, activating killer T cells, and the like. Killer T cells target those infected cells with specific antigenic information and kill them by releasing cytotoxins. T cells, like B cells, also use a detector called a T cell receptor to recognize specific antigens.

Recognition of an antigen is accomplished when the B cell receptor or T cell receptor is able to bind to certain portions of the antigen. These moieties capable of being bound are referred to as antigenic determinants or epitopes. Epitope identification has important significance for developing SARS-CoV-2 vaccine and monitoring epidemic situation.

Disclosure of Invention

According to one aspect of the present invention, there is provided an epitope peptide having an amino acid sequence selected from the group consisting of:

(1) the amino acid sequence of the epitope peptide is shown in SEQ ID NO. 1-4;

(2) the amino acid sequence of the epitope peptide is formed by substituting, deleting, inserting and/or adding 1 or more amino acid sequences of the sequence shown by SEQ ID NO. 1-4.

In a specific embodiment of the invention, the amino acid sequence of the epitope peptide is shown in SEQ ID NO. 1-4. Preferably, the amino acid sequence of the epitope peptide is shown as SEQ ID NO. 1.

The epitope peptide of the present invention can be produced by conventional methods for synthesizing various peptides. For example, they can be prepared by organic chemical synthesis such as solid phase peptide synthesis, or by recombinant DNA techniques by preparing nucleic acids encoding peptides. Alternatively, the peptide can be synthesized using a commercially available chemical synthesis apparatus (e.g., a peptide synthesis apparatus available from Applied Biosystems).

According to another aspect of the present invention, there is provided a nucleic acid encoding the epitope peptide as defined above.

Nucleic acids encoding epitope peptides are important for producing epitope peptides in a host using genetic recombination techniques. Because codons for amino acids vary in codon usage frequency among hosts, it is preferable to alter codons for amino acids to fit the codon usage frequency of the producing host. The nucleic acid encoding the epitope peptide is also important in the case of vaccines, and may be delivered in the form of naked nucleic acid, or may be delivered using an appropriate viral or bacterial vector. Suitable bacterial vectors are bacteria of the Salmonella subspecies. Suitable viral vectors are, for example: retrovirus vector, EBV vector, pox virus vector, Sendai virus vector, lentivirus vector.

According to a further aspect of the invention, there is provided a recombinant vector comprising a nucleic acid as hereinbefore described.

The vector may in particular be selected from pcDNA; pTT (Durocher et al, Nucleic Acids Research 2002, Vol 30, No. 2); pTT3 (pTT with additional multiple cloning sites); pEFBOS (Mizushima, S. and Nagata, S., (1990) Nucleic Acids Research Vol 18, No. 17); pBV; pJV and pBJ.

According to a further aspect of the invention, there is provided a host cell comprising a nucleic acid as hereinbefore described or into which a recombinant vector as hereinbefore described has been introduced.

Host cells are transformed with the vectors disclosed herein. Preferably, the host cell is a prokaryotic cell. More preferably, the host cell is e. In a related embodiment, the host cell is a eukaryotic cell. Preferably, the eukaryotic cell is selected from the group consisting of a protist cell, an animal cell (such as mammalian cell, avian cell and insect cell), a plant cell and a fungal cell. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS; or a fungal cell, such as a yeast cell, e.g., Saccharomyces cerevisiae; or insect cells such as Sf 9.

The recombinant vector may be transformed, transduced or transfected into a host cell by methods conventional in the art, such as chemical transformation by calcium chloride, high-voltage shock transformation, preferably shock transformation.

The epitope peptide of the present invention can be isolated and purified from a recombinant host cell using a method commonly used in the art. For example, the epitope peptide can be purified by centrifugation of the culture medium and the recombinant host cells, high-pressure homogenization for cell disruption, centrifugation for cell debris removal, and affinity chromatography. For the isolation and purification of the resulting product, purity identification can be performed using a method commonly used in the art. For example, Coomassie blue method, Kjeldahl method, biuret method, lowry method, ultraviolet absorption method, affinity chromatography, antigen-antibody method, electrophoresis (for example, sodium dodecyl sulfate polyacrylamide gel electrophoresis), sedimentation analysis, diffusion analysis, isotachy method, protein mass spectrometry, and the like.

According to still another aspect of the present invention, there is provided a vaccine comprising the epitope peptide as described above as an effective ingredient.

The epitope peptide of the present invention can be used as a peptide vaccine in active immunotherapy. That is, a vaccine prepared by administering a vaccine containing the epitope peptide of the present invention to a patient can proliferate T cells in vivo, and is effective for prevention and treatment of infection. The epitope peptide used may be only 1 kind, or 2 or more kinds of peptides may be combined and mixed depending on the purpose of use of the vaccine.

According to still another aspect of the present invention, there is provided a vaccine comprising the aforementioned nucleic acid as an active ingredient.

According to still another aspect of the present invention, there is provided a vaccine comprising the aforementioned recombinant vector as an effective ingredient.

The nucleic acid encoding the epitope peptide of the present invention can be used in DNA vaccines, recombinant viral vector vaccines, and the like in active immunotherapy. The nucleic acid sequence encoding the epitope peptide is preferably altered to a codon usage frequency suitable for a recombinant vaccine, the host producing the recombinant viral vaccine.

According to still another aspect of the present invention, there is provided a vaccine comprising as an active ingredient antigen-presenting cells that present the epitope peptide described above to HLA.

The antigen-presenting cells presenting the T-cell epitope peptide of the present invention can be used as a vaccine in active immunotherapy. Antigen presenting cells presenting T cell epitope peptides refer to:

(1) epitope peptide-pulsed antigen-presenting cells obtained by mixing antigen-presenting cells with epitope peptide in an appropriate culture solution for 30 minutes to 1 hour;

(2) cells in which epitope peptide is presented on antigen-presenting cells by gene transfer or the like using nucleic acid encoding epitope peptide

(3) An artificial antigen-presenting cell having an artificially prepared antigen-presenting ability.

Antigen-presenting cells are, for example, dendritic cells, B cells, macrophages, certain T cells, and the like, express HLA molecules to which the peptides can bind on the cell surface, and have CTL stimulatory ability.

The epitope peptide of the present invention or a vaccine comprising antigen-presenting cells presenting the epitope peptide can be prepared by a method known in the art. For example, as the vaccine, there are an injection, a solid preparation, and the like containing the epitope peptide of the present invention as an active ingredient. The epitope peptide may be formulated in a neutral or salt form, and examples of the pharmaceutically acceptable salt include inorganic salts such as hydrochloric acid and phosphoric acid, and organic acids such as acetic acid and tartaric acid. In addition, the antigen-presenting cells presenting the epitope peptide of the present invention may be mixed with pharmaceutically acceptable excipients compatible with the activity of the peptide or the cells, such as water, saline, glucose, ethanol, glycerol, DMSO (dimethyl sulfoxide), and other adjuvants, or a combination thereof. Further, if necessary, an auxiliary agent such as albumin, a wetting agent, an emulsifier, or the like may be added.

The vaccine of the present invention can be administered by parenteral administration or oral administration, and generally, parenteral administration is preferred. Examples of parenteral administration include nasal administration, injections such as subcutaneous injection, intramuscular injection and intravenous injection, and suppositories. For oral administration, a mixture with excipients such as starch, mannitol, lactose, magnesium stearate, cellulose, and the like may be prepared.

The vaccines of the present invention are administered in a therapeutically effective amount. The dose to be administered depends on the subject to be treated and the immune system, and the necessary dose is determined at the discretion of the clinician. The administration interval may be set according to the object or purpose.

According to a further aspect of the present invention, there is provided an antibody prepared from the epitope peptide described above.

The term "antibody" means an immunoglobulin molecule consisting of 4 polypeptide chains, two heavy (H) chains and two light (L) chains. The chains are typically linked to each other by disulfide bonds. Each heavy chain consists of the variable region of the heavy chain (abbreviated herein as HCVR or VH) and the constant region of the heavy chain. The heavy chain constant region consists of three regions, CH1, CH2, and CH 3. Each light chain consists of a variable region of the light chain (herein abbreviated as LCVR or VL) and a constant region of the light chain. The light chain constant region consists of a CL region. The VH and VL regions may be further divided into hypervariable regions known as Complementarity Determining Regions (CDRs) and alternating conserved regions known as Framework Regions (FRs). Thus, each VH and VL region consists of three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. This structure is well known to those skilled in the art.

The antibodies of the present invention can be prepared using conventional techniques in the art, and methods for preparing antibodies commonly used in the art include: (1) mouse/rabbit based hybridoma technology; (2) antibody screening techniques based on phage antibody display libraries; (3) screening techniques based on monoclonal antibody libraries.

According to still another aspect of the present invention, there is provided a passive immunotherapy agent against a novel coronavirus, which comprises a novel coronavirus-specific T cell obtained by stimulating peripheral blood lymphocytes with the epitope peptide as described above or an antigen-presenting cell that presents the epitope peptide to HLA.

According to yet another aspect of the present invention, there is provided a method for quantifying novel coronavirus-specific T cells, comprising:

stimulating peripheral blood from a subject with an epitope peptide as described previously;

obtaining novel coronavirus-specific T cells produced by the previous steps;

determining the production of cytokines and/or chemokines and/or cell surface molecules by the obtained T cells.

According to still another aspect of the present invention, there is provided a novel induction method of coronavirus-specific T cells, which comprises using the epitope peptide as described above; preferably, the method comprises contacting the antigen presenting cell with an epitope peptide as described above.

According to a further aspect of the present invention, there is provided a method for producing a novel coronavirus-specific T cell, which comprises using the epitope peptide as defined above, preferably the method comprises contacting a peripheral blood mononuclear cell with the epitope peptide as defined above.

According to still another aspect of the present invention, there is provided a kit for inducing T cells, comprising the epitope peptide as described above as a constituent.

The kit comprises the epitope peptide, or a detection reagent of the novel coronavirus, or a detection test paper of the novel coronavirus.

The detection reagent for the novel coronavirus comprises the epitope peptide. For example, the reagent is a magnetic bead reagent, and the epitope peptide is coated on the magnetic bead.

The novel coronavirus detection test paper comprises a substrate and the epitope peptide arranged on the substrate.

Further, the kit can also comprise at least one of a sample diluent, a coating solution, a sealing solution, a washing solution and a developing solution.

Further, the sample diluent includes carbonate buffer, PBS buffer, TBS buffer.

Specifically, the coating solution may include carbonate buffer, PBS buffer, TBS buffer. It will of course be appreciated that in other embodiments, the coating fluid may be other coating fluids commonly found in the art.

Specifically, the blocking solution can comprise a PBS solution containing 1-3% (v/v) BSA, a Casein solution containing 1-3% (v/v) BSA, and a 10% (v/v) horse serum solution. It will of course be appreciated that in other embodiments, the confining liquid may be other confining liquids commonly found in the art.

Specifically, the washing solution comprises PBST containing 0.05-0.2% (v/v) Tween-20, TBS containing 0.05-0.2% (v/v) Tween-20, and 10% Triton. It will of course be appreciated that in other embodiments, the washing solution may be other washing solutions common in the art.

The use method of the kit comprises the following steps:

mixing a sample to be detected with the epitope peptide coated on the solid phase carrier, incubating, adding a detection antibody, continuing incubating, and detecting to obtain a detection result.

In particular, the detection antibody is capable of binding to a novel coronavirus antibody and carries a detection label. The detection label may be any one of an enzyme label, a fluorescein label, a biotin label, and a colloidal gold label.

Specifically, the developing solution is selected according to the type of the label of the detection antibody.

According to a further aspect of the present invention, there is provided the use of an epitope peptide as hereinbefore described for the preparation of a novel coronavirus vaccine.

According to a further aspect of the present invention, there is provided the use of an epitope peptide as described hereinbefore for the preparation of novel coronavirus specific T cells.

According to a further aspect of the invention, there is provided the use of an epitope peptide as hereinbefore described in the manufacture of a product for monitoring the evolution of a novel coronavirus.

According to a further aspect of the present invention, there is provided use of the epitope peptide as defined above in the preparation of a kit for inducing T cells.

According to a further aspect of the present invention, there is provided the use of an epitope peptide as hereinbefore described for the preparation of antibodies against a novel coronavirus.

According to a further aspect of the present invention, there is provided the use of an epitope peptide as hereinbefore described in the manufacture of a kit for the detection of a novel coronavirus infection.

The kit is as defined above.

According to a further aspect of the invention there is provided the use of a nucleic acid as hereinbefore described in the preparation of a novel coronavirus vaccine.

According to a further aspect of the present invention, there is provided the use of a recombinant vector as hereinbefore described in the preparation of a novel coronavirus vaccine.

According to a further aspect of the present invention, there is provided use of a novel coronavirus-specific T cell obtained by stimulating peripheral blood lymphocytes with the epitope peptide as described above or an antigen-presenting cell which presents the epitope peptide to HLA, for the preparation of a passive immunotherapy agent for a novel coronavirus.

Drawings

FIG. 1 shows a T cell epitope peptide score plot;

FIG. 2 shows a graph of flow results of detection of CD44 and CD62L expression using a flow cytometer;

FIG. 3 shows a statistical plot of the variation in total cell number and T cell number in lymph nodes;

FIG. 4 shows a statistical plot of the variation in total cell number and T cell number in the spleen.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 epitope prediction

T-cell epitopes of SARS-CoV-2N protein (NCBI access number: YP-009724397.2) were predicted using the IEDB resource Class I immunology tool, which analyzed 600 immunogenic, 181 non-immunogenic 9mer peptides, summarizing two features. The first characteristic is that the peptide segment rich in phenylalanine, isoleucine and tryptophan has strong immunogenicity, while the peptide segment rich in serine, lysine and methionine has weak immunogenicity. Second, amino acids 4-6 of the 9mer peptide play an important role in the immunogenicity of the peptide fragment. The protein sequence was broken down into a series of 9mer peptides, with 8mer overlaps of 9mer peptides adjacent to each other. And the first, second and C-terminal amino acids of the 9mer peptide are masked. The score for each 9mer peptide was then calculated and the 4 highest scoring 9mer peptides were selected. See figure 1 and table 1. The fragments 12-20, 49-57,104-112,127-135 are found to have higher scores, wherein the fragments 127-135 have the highest score.

TABLE 19 basic information on mer peptides

Example 2 epitope peptide functional validation

1. Immunization of mice

Balb/c female mice, 8 weeks old, were purchased from Witongli Hua. The 9mer peptide (SEQ ID NO.1-4) was synthesized by Baisheng, Beijing Sai, and had a purity of 90% or more. Freund's complete adjuvant was purchased from Sigma. Pertussis toxin was purchased from Merck corporation. Peptides were dissolved at 2mg/ml in PBS, and the peptides were mixed completely with Freund's complete adjuvant (1: 1) in water-in-oil, 100. mu.g of peptide was immunized subcutaneously per mouse, and the control group was not antigen-treated. All mice were given an intraperitoneal injection of pertussis toxin at 0h, 24h after immunization, 500ng/0.2ml per mouse.

2. Flow cytometry

Preparation of FACS washing solution: 20mL fetal bovine serum and 1mL 10% NaN3 were added to 1L PBS solution, mixed well and stored at 4 ℃ until needed.

Counting after taking out spleen or lymph node cells of the mice, taking 1 × 10 of each sample⁶The cells were resuspended by adding 1mL of FACS wash, centrifuging at 6000rpm for 30s, discarding the supernatant, and adding 100. mu.L of FACS wash. Naked cells and a single label of different fluorescent channel antibodies were set. According to the following steps of 1: 100, adding fluorescent antibody, adding no antibody to naked cells, and adding only antibody of the corresponding channel to single label. Staining at 4 ℃ for 30min in the dark. 1mL FACS wash resuspended cells, centrifuged at 6000rpm for 30s, the supernatant discarded, 200. mu.L of 1% paraformaldehyde solution was added to each tube to immobilize the cells, and the cells were tested on the machine within a week. All flow antibodies were purchased from Biolegend.

3. Results

In this study, large female Balb/c mice were immunized subcutaneously for 8 weeks after epitope peptide synthesis, and draining Lymph Nodes (LN) and Spleen (SP) were removed 7 days later, both of which were found to be swollen, and cell counts showed an increase in total cell mass. Flow staining of CD4, CD8, CD44, CD62L resulted in an increase in both CD4+ and CD8+ T cell numbers. In addition, the expression conditions of CD44 and CD62L are analyzed, and the result shows that the fragment 127-135 has the strongest effect, so that the CD44 has the highest effect^lowCD62L^hi

The population ratio decreases. Other peptides do not affect CD44, CD62L expression in this short time, but because of the increased numbers of CD4+ and CD8+ T cells, both activated CD4+ and CD8+ T cells are increased. See figures 2-4 for results.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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Claims

1. An epitope peptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

2. A nucleic acid encoding the epitope peptide of claim 1.

3. A recombinant vector comprising the nucleic acid of claim 2.

4. A host cell comprising the nucleic acid of claim 2 or into which the recombinant vector of claim 3 has been introduced.

5. A vaccine comprising any one of:

(1) comprising the epitope peptide according to claim 1 as an active ingredient;

(2) comprising the nucleic acid according to claim 2 as an active ingredient;

(3) comprising the recombinant vector according to claim 3 as an effective ingredient;

(4) an antigen-presenting cell that presents the epitope peptide according to claim 1 to HLA, as an active ingredient.

6. A passive immunotherapy agent against a novel coronavirus, comprising a novel coronavirus-specific T cell obtained by stimulating peripheral blood lymphocytes with the epitope peptide of claim 1 or an antigen-presenting cell that presents the epitope peptide to HLA.

7. A method for non-therapeutic purposes, the method comprising any one of:

(1) a method for quantifying novel coronavirus-specific T cells, comprising:

stimulating peripheral blood from a subject with the epitope peptide of claim 1;

obtaining novel coronavirus-specific T cells produced by the previous steps;

determining cytokines and/or chemokines and/or cell surface molecules produced by the obtained T cells;

(2) a novel induction method of coronavirus-specific T cells, comprising administering the epitope peptide of claim 1;

(3) a method for producing a novel coronavirus-specific T cell, which comprises using the epitope peptide according to claim 1.

8. The method according to claim 7, wherein the method in (2) comprises contacting an antigen-presenting cell with the epitope peptide according to claim 1.

9. The method according to claim 7, wherein the method in (3) comprises contacting peripheral blood mononuclear cells with the epitope peptide according to claim 1.

10. A kit for inducing T cells, comprising the epitope peptide according to claim 1 as a constituent element.

11. An application, the application comprising any one of:

(1) use of the epitope peptide of claim 1 for the preparation of a novel coronavirus vaccine;

(2) use of the epitope peptide of claim 1 for the preparation of novel coronavirus-specific T cells;

(3) use of the epitope peptide of claim 1 for the preparation of a product for monitoring a novel coronavirus;

(4) use of the epitope peptide of claim 1 for the preparation of a kit for inducing T cells;

(5) use of the epitope peptide of claim 1 for the preparation of antibodies against a novel coronavirus;

(6) use of the epitope peptide of claim 1 for the preparation of a kit for the detection of a novel coronavirus infection;

(7) use of the nucleic acid of claim 2 for the preparation of a novel coronavirus vaccine;

(8) use of the recombinant vector of claim 3 for the preparation of a novel coronavirus vaccine;

(9) use of a novel coronavirus-specific T cell obtained by stimulating peripheral blood lymphocytes with the epitope peptide of claim 1 or an antigen-presenting cell that presents the epitope peptide to HLA, for the preparation of a passive immunotherapy agent for a novel coronavirus.