Tetanus vaccine and enzyme-linked immunospot assay method thereof

Abstract

This invention discloses a tetanus vaccine and its enzyme-linked immunosorbent assay (ELISpot) analysis method. The tetanus vaccine of this invention is designed based on screened tetanus antigen peptides, which contain any of the amino acid sequences shown in SEQ ID NO:1-SEQ ID NO:61. Addressing the technical problem of the lack of specific T-cell immune response detection methods for existing tetanus vaccines, this invention designs an overlapping peptide library covering the entire sequence of tetanus toxoid, screening out dominant peptides that can effectively stimulate T-cell responses, significantly improving the sensitivity and specificity of detection. This method optimizes the processing flow of PBMCs in the test samples, establishes a quantitative analysis standard based on IFN-γ secretion, and achieves accurate assessment of the T-cell immune response induced by the tetanus vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a tetanus vaccine and its enzyme-linked immunospot assay technique. Background Technology

[0002] With the widespread application of enzyme immunoassay technology in the fields of medicine and biology, new breakthroughs have been achieved in the in vitro detection of various cytokines and antibodies. In studying immune response mechanisms, enzyme-linked immunosorbent assay (ELISA) was previously commonly used to detect free cytokines (CK) or antibodies in body fluids. However, due to the different half-lives of free circulating antibodies or CK, they are continuously metabolized or bound to target organs in body fluids, failing to accurately reflect the levels of antibodies and CK in vivo. Enzyme-linked immunospot (ELISpot) technology, on the other hand, can perform cellular monitoring at the single-cell level. In the mid-1980s, Sedgwick, Holt, and Czerk in Skye, based on the basic principles of ELISA, established a solid-phase ELISpot technique for the in vitro detection of specific antibody-secreting cells and CK-secreting cells. Due to its high specificity and sensitivity, it is currently widely used both domestically and internationally.

[0003] In recent years, due to the enormous impact of SARS-CoV-2 on human life, researchers both domestically and internationally have made significant strides in the development of inactivated and detoxified vaccines. Inactivated and detoxified vaccines (such as tetanus vaccines, which are detoxified tetanus toxoids) involve directly inactivating the virus or extracting and detoxifying the toxin before injecting it into the body to trigger an immune response. ELISpot technology has been widely used to detect vaccine efficacy, for example, to analyze and evaluate antigen-specific T-cell immune responses detected from PBMCs extracted from peripheral blood.

[0004] Tetanus vaccines are primarily tetanus toxoids, which are inactivated from tetanus toxin using formaldehyde. Theoretically, tetanus toxoids provide all neutralizing antibodies against tetanus toxin, but studies have shown that they cannot produce all neutralizing antibodies. This indicates that formaldehyde inactivation may lead to major conformational changes in tetanus toxoids, reducing their immunogenicity and efficacy. Currently, the production process for tetanus toxoids in China often involves detoxification followed by purification. The drawback of this process is that culture medium components in the toxin can easily cross-link with toxin molecules through formaldehyde during detoxification, making purification difficult, time-consuming, and lengthy. The acellular DPT combined vaccine commonly administered in my country is a second-generation DPT vaccine. Due to its co-passivation process, the effective antigen components cannot be precisely quantified, posing challenges to production and quality control. Therefore, it is necessary to develop a safe, effective, and convenient vaccine to prevent tetanus poisoning. Furthermore, the development of ELISpot analysis technology for vaccine products will play a crucial role in monitoring the efficacy of such products. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the lack of enzyme-linked immunospot analysis technology for tetanus vaccines in the existing technology, to determine the optimal experimental conditions and to obtain specific stimulating peptides.

[0006] Based on an enzyme-linked immunospot (ELISA) analysis platform, the inventors designed and synthesized 189 polypeptides with 9 overlapping amino acids and a length of 15 amino acids, according to the tetanus toxoid protein sequence. A complete peptide library containing these 189 polypeptides was constructed, and the 189 polypeptides were divided into 30 peptide pools to form a matrix. Four polypeptides could not be obtained due to purification difficulties, so their lengths were shortened from 15 amino acids to 10 amino acids: CALSVSSVAQLDLSS (SEQ ID NO: 62) was deleted, and the actual polypeptide sequence used was SSVAQLDLSS; LITIAASNNITVELW (SEQ ID NO: 63) was deleted, with amino acid residues positions 1-3 and 13-14 being deleted, and the actual polypeptide sequence used was IAASNNITVE; SNNITVELWTKVNSA (SEQ ID NO: 64) was deleted, and the actual polypeptide sequence used was SNNITVELWT; ITTVSKLFEVEYVNK (SEQ ID NO: 63) was deleted, with amino acid residues positions 11-15 being deleted, and the actual polypeptide sequence used was SNNITVELWT; ITTVSKLFEVEYVNK (SEQ ID NO: 64) was deleted, with amino acid residues positions 11-15 being deleted, and the actual polypeptide sequence used was SNNITVELWT; The first to fifth amino acid residues of 65) are actually used in the polypeptide sequence KLFEVEYVNK. By extracting PBMCs from immunoassay samples, the extracted PBMCs are stimulated by a peptide library or matrix peptide pool to detect the immune response, establishing a complete enzyme-linked immunospot assay technique for tetanus vaccines and identifying the dominant polypeptide.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0008] A first aspect of the present invention provides a tetanus antigen peptide comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-SEQ ID NO: 61.

[0009] In some embodiments of the present invention, the tetanus antigen peptide comprises an amino acid sequence as shown in any of SEQ ID NO: 3, 4, 41, 42, 45, 53, 56, 57 and 60.

[0010] A second aspect of the present invention provides a pharmaceutical composition comprising the tetanus antigen peptide as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipients.

[0011] A third aspect of the present invention provides a tetanus vaccine comprising, as a first aspect of the present invention, a tetanus antigen peptide, or as a second aspect of the present invention, a pharmaceutical composition as an immunogenic component.

[0012] The fourth aspect of the present invention provides the use of a tetanus antigen peptide as described in the first aspect of the present invention, or a pharmaceutical composition as described in the second aspect of the present invention, in the preparation of a tetanus vaccine.

[0013] The fifth aspect of this invention provides a method for screening tetanus antigen peptides based on ELISPOT, the method comprising:

[0014] (1) Construct a complete peptide library of antigens;

[0015] (2) Rats were immunized with a complete peptide library to obtain whole blood PBMC samples;

[0016] (3) IFN-γ ELISpot assay was performed on whole blood PBMC samples to evaluate the cellular immune response level of each antigen peptide in the whole peptide library;

[0017] For each reaction well, when 10 5 The antigen peptide is considered to be a cellular immune response when the number of spots produced by each PBMC cell is at least twice the number of spots in the negative control well.

[0018] In some embodiments of the present invention, (1) the complete peptide library is constructed by synthesizing all polypeptides of 15 amino acids in the tetanus toxoid protein sequence; wherein one polypeptide overlaps with at least another polypeptide by 9 amino acids.

[0019] In some embodiments of the present invention, in 2), the use of the whole peptide library includes immunization using the antigen peptides of the whole peptide library one by one, or designing a matrix to mix a specific number of single peptides to form a peptide pool for immunization based on the number of antigen peptides in the whole peptide library.

[0020] In some embodiments of the present invention, the antigen is tetanus toxoid.

[0021] In some embodiments of the present invention, when immunization is performed using antigen peptides from the full peptide library, each reaction well contains one antigen peptide.

[0022] When using a peptide pool for immunization, each of the reaction wells contains multiple antigenic peptides.

[0023] The sixth aspect of the present invention provides an antigenic peptide library of tetanus toxoid, the antigenic peptide library comprising any of the amino acid sequences shown in SEQ ID NO: 1-SEQ ID NO: 61.

[0024] In some embodiments of the present invention, the antigen peptide library is used in screening tetanus antigen peptides, preparing tetanus vaccines, and evaluating tetanus vaccines.

[0025] In some embodiments of the present invention, the evaluation of the tetanus vaccine includes evaluating the immunogenicity of the tetanus vaccine.

[0026] In this invention, the term "ELISPOT" is short for Enzyme-Linked Immunospot Assay, a technique for detecting cytokines or other secreted proteins at the single-cell level. This method is characterized by high sensitivity and specificity, and can quantitatively analyze the amount of cytokines secreted by a single cell.

[0027] "PBMC" is short for Peripheral Blood Mononuclear Cell, referring to the population of mononuclear cells present in peripheral blood, mainly including lymphocytes and monocytes. In this invention, PBMC samples are obtained by isolating them from whole blood of rats for subsequent testing.

[0028] "IFN-γ" is short for Interferon-gamma, an important cytokine secreted by activated T lymphocytes and natural killer cells. In this invention, the strength of antigen-specific T cell immune responses is assessed by detecting the level of IFN-γ secreted by PBMCs.

[0029] A "peptide pool" refers to a mixture formed by combining multiple individual peptides according to a specific design scheme. In this invention, by designing a matrix to mix a specific number of single peptides to form a peptide pool, screening efficiency can be improved.

[0030] This invention establishes an Elispot analysis technique for tetanus vaccines by optimizing experimental conditions, identifies dominant peptides, and provides a reference for establishing Elispot methods for similar vaccines.

[0031] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0032] The reagents and raw materials used in this invention are all commercially available.

[0033] The positive and progressive effects of this invention are as follows: an enzyme-linked immunospot assay (ELISA) for tetanus vaccine was established, and nine specific peptides that can activate rat T-cell immune responses were screened from a complete peptide library of tetanus toxoid. By optimizing the PBMC processing technology and establishing quantitative analysis standards for IFN-γ secretion, accurate detection of T-cell immune responses to tetanus vaccine was achieved. Attached Figure Description

[0034] Figure 1 This study aims to assess the T-cell immune response levels in male animals after immunization under different peptide pool stimulation conditions. The results show the PBMC spot count report value (number of peptide pool stimulation spots - number of negative control spots) for the low-dose group animals.

[0035] Figure 2 This study aimed to assess the T-cell immune response levels in female animals after immunization under different peptide pool stimulation conditions. The results showed the PBMC spot count report value (number of peptide pool stimulation spots - number of negative control spots) for the high-dose group animals. Detailed Implementation

[0036] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0037] Example 1: Establishment of Elispot analysis technology for tetanus vaccine

[0038] 1. Experimental Procedure

[0039] 1.1 Animal Immunization and Grouping

[0040] Tetanus vaccine immunization grouping: SPF-grade SD rats were randomly divided into 3 groups (n=10, half male and half female). Each rat was immunized via intramuscular injection. The tetanus vaccine concentration was 88.6 IU / mL. Group 1 was the negative control group, immunized by intramuscular injection of 0.5 mL / rat of 0.9% sodium chloride injection; Group 2 was the low-dose group, immunized by intramuscular injection of 0.25 mL / rat; Group 3 was the high-dose group, immunized by intramuscular injection of 0.5 mL / rat. Blood samples were collected on D1 before administration, D14, and D28, where D represents the day.

[0041] 1.2 Establishment of Elispot analytical technique for tetanus vaccine products

[0042] a. Peptide pool mixing scheme: Based on the tetanus toxoid protein sequence, 189 peptides with 9 overlapping amino acids and a length of 15 amino acids were designed and synthesized. Among them, 4 peptides were replaced with 10 amino acid peptides. The synthesized peptides were dissolved in 100 μL LDMSO (Solarbio, Cat#3230821002) to obtain a complete peptide library reconstitution solution with a concentration of 1 mg / mL, and a complete peptide library containing 189 peptides was constructed.

[0043] b. Acquisition of PBMCs for immunoassay: Before drug administration at time point D1, approximately 1.2 mL of whole blood was collected from SD rats on D14 and D28, and the whole blood from female and male animals was mixed into one tube each.

[0044] (1) Take whole blood and dilute it with PBS-2%FBS at a ratio of 3:5 and mix well;

[0045] (2) Add an appropriate amount of sample density separation solution to a 15 mL high-efficiency centrifuge tube, centrifuge at 1500 rpm and 20°C for 1 min;

[0046] (3) Take the diluted whole blood from step 2 and slowly add it into a high-efficiency centrifuge tube (containing sample density separation solution), centrifuge at 2200 rpm, 20°C~25°C, 6 increments and 1 decrement for 30 min;

[0047] (4) Aspirate the supernatant (including the cloud layer) from each tube into a newly labeled 15 mL centrifuge tube, and gently invert it 3 to 5 times to obtain the PBMC sample;

[0048] (5) Add PBS-2%FBS to the newly labeled 15 mL centrifuge tube to make up to 10 mL, increase by 9, decrease by 9, centrifuge at 1500 rpm at room temperature for 10 min;

[0049] (6) Discard the supernatant and add 1 mL of cryopreservation solution (prepared by mixing FBS (gibco, Cat#: 2297786cp) and DMSO (Hangzhou Xiaoyou Biotechnology Co., Ltd., Cat#: 221017091) at a ratio of 9:1). After mixing, place it in a programmed cryopreservation box (bkmam, Cat#: 313228) and store at -80℃.

[0050] 1.3 ELISpot assay for tetanus vaccine-specific cellular immunity

[0051] The IFN-γ ELISpot level in PBMCs of SD rats was detected using Mabtech's ELISpot Plus: Rat IFN-γ (ALP) kit to evaluate the cellular immune response level to tetanus vaccine.

[0052] (1) Washing the plate: Under sterile conditions in the cell culture, take out the 96-well plate from the ELISpot Plus: Rat IFN-γ (ALP) kit, add 200 µL of sterile PBS to each well and wash the plate four times, then discard the liquid in the plate.

[0053] (2) Blocking: Under sterile conditions between cells, add 200 µL of 1640-10% FBS medium to each well, cover and incubate at room temperature for 30 min or more.

[0054] (3) Cell stimulation: The whole peptide library was used as a stimulant and diluted with 1640-10%FBS-1% penicillin-antibody medium (1640 medium (gibco, Cat#: 2537138), FBS (gibco, Cat#: N2603053p), penicillin-antibody (Shanghai Yuanpei Biotechnology Co., Ltd., Cat#: J121004)) (working concentration: 5 μg / mL / peptide chain). 100 μL of working solution was added to the wells, followed by 100 μL of PBMC cell suspension (1×10⁻⁶ cells / well). 6 Cells / mL), the concentration of DMSO in the negative control was consistent with the final concentration in the experimental group, and 20 ng / mL + 0.4 μg / mL (PMA + Ionomycin) served as the positive control. Incubation was performed at 37℃ and 5% CO2 for 18 h ± 0.5 h;

[0055] (4) Add Biotin working solution: Add 100 µL of Anti-IFN-γmAb (7-B6-1) and biotin working solution to each well, cover the well, and incubate at room temperature for 2 h ± 10 min;

[0056] (5) Add ALP working solution: Add 100 µL of Streptavidin-ALP diluent to each well, cover and incubate at room temperature for 1 h ± 10 min;

[0057] (6) Color development: Filter the BCIP / NBT-plus substrate for ELISpot through a 0.45 µm filter membrane, and add 100 µL of BCIP / NBT-plus substrate for ELISpot to each well until obvious spots appear;

[0058] (7) Reading and calculation: Use the ELISpot Reader (CTL) to read the spot plate and calculate the number of PBMC cells secreting IFN-γ per 100,000 PBMC cells.

[0059] (8) Positive criterion: 10 5 The number of spots produced by each PBMC cell was at least twice the number of spots in the negative control well.

[0060] 2. Experimental Results

[0061] Following drug administration, both the low- and high-dose groups showed stronger antigen-specific T-cell immune responses than the negative control group. The negative control group exhibited negative antigen-specific T-cell immune responses (or a low number of spots), while the low- and high-dose groups showed more pronounced antigen-specific T-cell immune responses. The number of spots between the detection well and the background well was used as the reporting value. The specific results are shown in Table 1 below.

[0062] Table 1 Reported values ​​for each group of animals

[0063]

[0064] The above results indicate that the established ELISpot analysis technique can be used for the evaluation of tetanus vaccines.

[0065] Example 2: Screening of Advantageous Peptides

[0066] A matrix was designed based on the total number of peptides in each peptide library. An appropriate number of single peptides were mixed together to form peptide pools, ensuring consistent concentration for each peptide. 189 peptides were divided into 30 pools: pools 1-21 each contained 9 peptides, and pools 22-30 each contained 21 peptides. Each peptide pool served as a stimulant for IFN-γ-ELISpot detection. Each peptide appeared once in each sub-pool; therefore, cross-referencing the pools with positive reactions horizontally and vertically based on the detection results could identify single peptides that might produce a positive reaction. Detailed matrix scheme is shown in Table 2 below.

[0067] Table 2. Peptide library matrix design table

[0068]

[0069] The different peptide pools were tested according to the method established in Example 1, and the trend results are shown in [Figure 1]. Figure 1 and Figure 2 The detailed results are shown in Table 3. The above results indicate that, based on the judgment criteria, a pool of positive peptides that can be recognized by PBMC and the single peptides obtained by crossover can be screened out. The results are shown in Table 4. The single peptide numbers obtained by crossover are 3, 4, 66, 67, 75, 76, 165, 166, 174, 175, 183 and 184, respectively, and the corresponding peptide sequences are LGLTLVCALSVSSVA, SSVAQLDLSS, LTTPSPTKAKPLTTP, TKAKPLTTPTPKKPQ, TPSPTKAKPLTTPTP, AKPLTTPTPKKPQLL, LPTTSLTTPSPTKAKP, TPSPTKAKPLTTPTP, TSLTTPSPTKAKPLT, SPTKAKPLTTPTPKK, LTTPSPTKAKPLTTP, TKAKPLTTPTPKKPQ, and the corresponding sequence numbers are SEQ ID NO: 3, 4, 45, 53, 41, 42, 60, 41, 56, 57, 45 and 53.

[0070] Table 3. Immune response results of animals in low- and high-dose groups after administration of different peptide pool stimulation drugs.

[0071]

[0072] Note: Negative values ​​in the report are all counted as 0.

[0073] Table 4. Positive peptide library obtained from screening

[0074]

[0075] The specific embodiments described above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements or substitutions without departing from the principle of the present invention, and these improvements or substitutions should also be considered within the scope of protection of the present invention.

[0076] Appendix: Sequence List

[0077]

[0078]

[0079]

[0080]

[0081]

Claims

1. A tetanus antigen peptide, characterized in that, The tetanus antigen peptide comprises an amino acid sequence as shown in any of SEQ ID NO: 1-SEQ ID NO:

61.

2. The tetanus antigen peptide as described in claim 1, characterized in that, The tetanus antigen peptide comprises an amino acid sequence as shown in any of SEQ ID NO: 3, 4, 41, 42, 45, 53, 56, 57 and 60.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the tetanus antigen peptide as described in claim 1 or 2, and a pharmaceutically acceptable carrier and / or excipient.

4. A tetanus vaccine, characterized in that, The tetanus vaccine comprises the tetanus antigen peptide as described in claim 1 or 2, or the pharmaceutical composition as described in claim 3, as an immunogenic component.

5. The use of a tetanus antigen peptide as described in claim 1 or 2, or a pharmaceutical composition as described in claim 3, in the preparation of a tetanus vaccine.

6. A method for screening tetanus antigen peptides based on ELISPOT, characterized in that, The method includes: (1) Construct a complete peptide library of antigens; (2) Rats were immunized with a complete peptide library to obtain whole blood PBMC samples; (3) IFN-γ ELISpot assay was performed on whole blood PBMC samples to evaluate the cellular immune response level of each antigen peptide in the whole peptide library; For each reaction well, when 10 5 The antigen peptide is considered to be a cellular immune response when the number of spots produced by each PBMC cell is at least twice the number of spots in the negative control well.

7. The method as described in claim 6, characterized in that, In (1), the complete peptide library is constructed by synthesizing all polypeptides of 15 amino acids in length from the tetanus toxoid protein sequence; in the polypeptides, one polypeptide overlaps with at least another polypeptide by 9 amino acids; and / or, (2) The use of the full peptide library includes using the antigen peptides of the full peptide library one by one for immunization, or designing a matrix to mix a specific number of single peptides to form a peptide pool for immunization based on the number of antigen peptides in the full peptide library. And / or, the antigen is tetanus toxoid.

8. The method as described in claim 7, characterized in that, When immunization is performed using antigen peptides from the full peptide library, each reaction well contains one antigen peptide. When using a peptide pool for immunization, each of the reaction wells contains multiple antigenic peptides.

9. A library of antigenic peptides for tetanus toxoid, characterized in that, The antigen peptide library contains amino acid sequences as shown in any of SEQ ID NO: 1-SEQ ID NO:

61.

10. The application of the antigen peptide library as described in claim 9 in screening tetanus antigen peptides, preparing tetanus vaccines, and evaluating tetanus vaccines; Preferably, the evaluation of the tetanus vaccine includes evaluating the immunogenicity of the tetanus vaccine.

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