A composite antigen polypeptide and a hydatid disease vaccine

Abstract

The present application belongs to the technical field of immunobiology, and particularly relates to a kind of complex antigen polypeptide and echinococcosis vaccine, wherein the complex antigen polypeptide includes amino acid sequence with more than 80% homology with the amino acid sequence of MHC-II P1, MHC-II P2 or MHC-I P3.The present application connects multiple antigen epitopes using a linker, successfully constructs a complex polypeptide FL46, and enhances the immunogenicity of the epitope;The present application forms a FL46 polypeptide Freund's adjuvant vaccine by combining Freund's adjuvant with the polypeptide FL46, and verifies the protective effect of the vaccine on mice with granular echinococcosis, and achieves a significant cyst reduction effect.

Description

Technical Field

[0001] This invention belongs to the field of immunobiology, and in particular relates to a complex antigen polypeptide and a vaccine for echinococcosis. Background Technology

[0002] Echinococcosis, also known as echinococcosis, is a serious zoonotic parasitic disease caused by the larvae of the Echinococcus tapeworm parasitizing the liver, lungs, spleen, and other organs of humans and animals. It severely endangers human health and the development of animal husbandry. Echinococcosis is mainly divided into cystic echinococcosis (CE) and alveolarechinococcosis (AE), with cystic echinococcosis being the most common and having a high morbidity and mortality rate.

[0003] Echinococcus granulosus larvae primarily parasitize organs such as the liver, lungs, and spleen, causing harm to humans and animals through mechanical compression, allergic reactions, and toxic effects, with the liver being the most severely affected organ. Currently, apart from surgical removal of large, single cysts, which is relatively effective, there is still a lack of effective treatments for scattered, multiple, and smaller cysts of echinococcosis. Commonly used drugs such as albendazole and praziquantel have certain side effects, and prophylactic vaccination is an effective supplement to comprehensive prevention and control of echinococcosis. Research on Echinococcus granulosus vaccines has progressed from crude antigens, cell vaccines, recombinant protein vaccines, DNA vaccines, and peptide vaccines. Traditional vaccines prepared from Echinococcus granulosus secretions and metabolites are costly to produce and carry the risk of infection; recombinant protein vaccines, such as the commercially available Eg95, are expensive and require multiple immunizations; DNA vaccines may cause side effects such as autoimmune diseases or insertional mutations. Epitope vaccines have advantages such as flexible design, high specificity, no risk of infection, and ease of production and storage, representing a new direction in vaccine research. Multi-epitope vaccines can contain multiple dominant B or T cell antigenic epitopes of pathogens, which can simultaneously induce cellular and humoral immunity in the body, overcoming the shortcomings of limited immune protection from single antigens. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a complex antigenic polypeptide to solve the problem of limited immunoprotection by single antigens in the prior art. This invention also provides a vaccine for echinococcosis. This invention uses linkers to connect multiple antigenic epitopes, successfully constructing the complex polypeptide FL46, which enhances the immunogenicity of the epitopes. This invention combines Freund's adjuvant with polypeptide FL46 to construct the FL46 polypeptide Freund's adjuvant vaccine, and verifies the protective effect of this vaccine against mouse echinococcosis, achieving a significant reduction in cysts.

[0005] To achieve the above objectives and other related objectives,

[0006] In a first aspect, the present invention provides a complex antigen polypeptide comprising an amino acid sequence having more than 80% homology with the amino acid sequence of MHC-IIP1, MHC-IIP2 or MHC-IP3.

[0007] The amino acid sequence of the MHC-IIP1 is shown in SEQ ID NO:1;

[0008] The amino acid sequence of the MHC-IIP2 is shown in SEQ ID NO:2;

[0009] The amino acid sequence of the MHC-IP3 is shown in SEQ ID NO:3.

[0010] The major histocompatibility complex (MHC) is a highly polymorphic genome consisting of a group of chromosomes encoding proteins related to intercellular recognition and antigen presentation. In humans, the MHC is called the HLA complex. The complex epitope polypeptide FL46 prepared using this method exhibits high binding affinity and immunogenicity to both HLA-I (MHC-I) and HLA-II (MHC-II) molecules, and can be used in the preparation of Echinococcus granulosus vaccines.

[0011] In embodiments of the present invention, the complex antigen polypeptide includes an amino acid sequence having more than 80% homology with the amino acid sequences of MHC-IIP1, MHC-IIP2 and MHC-IP3.

[0012] In embodiments of the present invention, the complex antigen polypeptide comprises an amino acid sequence having more than 90% homology with the amino acid sequences shown in MHC-IIP1, MHC-IIP2 and MHC-IP3.

[0013] In embodiments of the present invention, the complex antigen polypeptide includes MHC-IIP1, MHC-IIP2 and MHC-IP3.

[0014] In embodiments of the present invention, the complex antigen polypeptide includes MHC-IIP1, MHC-IIP2 and MHC-IP3, wherein the number of MHC-IIP1 is at least two.

[0015] In embodiments of the present invention, the first MHC-IIP1 and the second MHC-IIP1 in the complex antigen polypeptide are linked by a first KKK linker, the second MHC-IIP1 and MHC-IIP2 are linked by a second KKK linker, and the MHC-IP3 and MHC-IIP2 are linked by a GGPPG linker.

[0016] In embodiments of the present invention, the amino acid sequence of the complex antigen polypeptide is shown in SEQ ID NO:4.

[0017] In a second aspect, the present invention provides the use of the above-mentioned complex antigenic polypeptide in the preparation of a vaccine against Echinococcus granulosus.

[0018] A third aspect of the present invention provides an echinococcosis vaccine, the echinococcosis vaccine comprising the above-mentioned complex antigen polypeptide.

[0019] In an embodiment of the present invention, the echinococcosis vaccine is a granular echinococcosis vaccine.

[0020] In an embodiment of the present invention, the Echinococcus granulosus vaccine comprises:

[0021] The above-mentioned complex antigen polypeptide; and

[0022] Freund's complete adjuvant or Freund's incomplete adjuvant.

[0023] As described above, the composite antigen polypeptide and echinococcosis vaccine of the present invention have the following beneficial effects:

[0024] (1) The composite antigen polypeptide of the present invention uses linkers to link three antigen polypeptides, which overcomes the disadvantage of limited immune protection of a single antigen and improves the immunogenicity of the vaccine.

[0025] (2) The complex antigen polypeptide of the present invention can simultaneously induce cellular immunity and humoral immunity in the body, promote the body to produce a large number of cytokines and antibodies, and improve the body's immune level.

[0026] (3) The polypeptide Freund's adjuvant vaccine of the present invention significantly reduces the size, number and weight of Echinococcus granulosus larvae after infection with Echinococcus granulosus, with a cyst weight reduction rate of (59.16±1.83)%, and has a significant protective effect against Echinococcus granulosus infection. Attached Figure Description

[0027] Figure 1 This describes the construction method of the Echinococcus granulosus complex epitope polypeptide FL46 in Example 1 of the present invention.

[0028] Figure 2 This is the HPLC analysis chromatogram of the composite epitope FL46 in Example 1 of the present invention.

[0029] Figure 3 This is an MS analysis chromatogram of the composite epitope FL46 from Example 1.

[0030] Figure 4This is Example 2 of the present invention, which uses the CCK8 assay to detect the proliferative effect of FL46 on BMDCs. BMDCs were stimulated with FL46 protein for 24 hours. The cell viability is statistically analyzed, with the horizontal axis representing the concentration of different FL46 proteins and the vertical axis representing cell viability. ns represents non-significant difference, and * represents significant difference, with the number of asterisks indicating the degree of significance.

[0031] Figure 5 This is from Example 2 of the present invention, where the CCK8 assay was used to detect the proliferative effect of FL46 on U937 cells. FL46 protein stimulated U937 cells for 24 hours. The x and y axes are the same as... Figure 3 .

[0032] Figure 6 Example 3 describes the mouse vaccine immunization regimen. Mice were randomly divided into a Freund's adjuvant group and a peptide Freund's adjuvant group. Mice in each group were subcutaneously injected with Freund's adjuvant and FL46 peptide Freund's adjuvant complex in their backs, respectively.

[0033] Figure 7 Example 4 describes the use of the MSD chip to detect the expression levels of serum cytokines in mice at different time points after immunization, including TNF-α, IL-2, IL-5, IL-12p70, and IL-10. Blood was collected from the orbital rim of mice, centrifuged, and the serum was sent to Unimicron Technology for analysis. In the statistical graph, ns represents non-significant differences, * represents significant differences, and the number of * indicates the degree of significance.

[0034] Figure 8 Example 5 shows the detection of serum IgG and IgG2a antibody titers in mice 2 weeks and 8 months after the last immunization using the indirect ELISA method. In the statistical graph, ns represents non-significant differences, and * represents significant differences. The number of ns represents the degree of significance.

[0035] Figure 9 This is the flowchart of mouse vaccine immunization and Echinococcus granulosus challenge infection in Example 3. Two weeks after three immunizations, the protoscolex was injected intraperitoneally for infection.

[0036] Figure 10 Example 6 shows the size and number of cysts in the abdominal cavity of mice observed with the naked eye.

[0037] Figure 11 Example 6 shows the weight of intraperitoneal cysts in mice. Detailed Implementation

[0038] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0039] Example 1

[0040] Chemical synthesis and characterization of the complex polypeptide FL46

[0041] Zhongtai Biochemical Co., Ltd. linked three T-cell epitope antigen peptides P1, P2, and P3 using appropriate linkers. The first KKK linker was used to link the first HLA-II (MHC-II) molecule-binding peptide P1 to the second HLA-II (MHC-II) molecule-binding peptide P2. The second KKK linker was used to link the second HLA-II (MHC-II) molecule-binding peptide P1 to the HLA-II (MHC-II) molecule-binding peptide P2. The GGPPG linker was used to link the HLA-I (MHC-I) molecule-binding peptide P3 to the aforementioned HLA-II (MHC-II) molecule-binding peptide P2, constructing a complex epitope peptide FL46. Figure 1 ).

[0042] The amino acid sequence of MHC-IIP1 is shown in SEQ ID NO:1, specifically FLQSEQLT.

[0043] The amino acid sequence of MHC-IIP2 is shown in SEQ ID NO:2, specifically AFIDTEVAAT.

[0044] The amino acid sequence of MHC-IP3 is shown in SEQ ID NO:3, specifically FEQEMATAA.

[0045] The amino acid sequence of the complex epitope polypeptide FL46 is shown in SEQ ID NO:4, specifically FLQSEQLTKKKFLQSEQLTKKKAFIDTEVAATGPGPGFEQEMATAA.

[0046] The purity and molecular weight of the complex epitope peptide FL46 were analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). HPLC results showed that the FL46 complex peptide exhibited a peak at 6.310 min, with a purity of 96.9%. Figure 2 The molecular weight of the FL46 complex peptide, as analyzed by MS, was 5044.8 g / mol. Figure 3 ).

[0047] Example 2

[0048] In vitro detection of the proliferative effect of the complex epitope peptide FL46 on BMDCs and U937 cell lines using the CCK8 assay.

[0049] (1) BMDC proliferation

[0050] ① Primary bone marrow-derived dendritic cells (BMDCs) were obtained by severing mice;

[0051] ② Collect BMDCs cultured to day 7 and adjust the cell concentration to 1×10⁻⁶. 6 Cells / mL, cultured in 96-well plates;

[0052] ③ Different concentrations (20, 40, 80, 100, 200 μg / mL) of FL46 complex peptide (eptopy peptide FL46) were added to the experimental groups. The negative control group was added with an equal volume of PBS and incubated at 37℃ in a 5% CO2 incubator for 24 h.

[0053] ④ Add CCK8 reagent and incubate in an incubator for 2–3 hours.

[0054] ⑤ The absorbance at 450 nm was measured using an ELISA reader to calculate cell viability.

[0055] (2) U937 proliferation

[0056] ① Adjust the density of U937 cells (monocyte cell line) to 1×10⁻⁶. 6 Cells / mL were seeded into 24-well plates;

[0057] ② Add 100 ng / mL of phorbol ester (PMA) to each well and incubate at 37°C in a 5% CO2 incubator for 48 h to induce the cells to transform into macrophages. Discard any cells that do not adhere to the cell wall.

[0058] ③ Add different concentrations (200, 250, 300, 350, 400, 450 and 500 μg / mL) of the compound polypeptide FL46 respectively;

[0059] ④ The remaining operations are the same as BMDCs.

[0060] The results showed that, compared with the PBS negative control group, there was no statistically significant difference in cell viability of primary BMDCs stimulated with different concentrations (20, 40, 80, 100, 200 μg / ml) of FL46 complex peptide for 24 h (P>0.05), indicating that FL46 complex peptide at concentrations below 200 μg / mL did not affect the viability of BMDCs and was non-toxic to BMDCs. Figure 4 ).

[0061] Stimulation of U937 cells with different concentrations (200, 250 μg / mL) of FL46 complex peptide for 24 h resulted in a slight increase in cell proliferation (P<0.05); however, stimulation of U937 cells with concentrations (300, 350, 400, 450, and 500 μg / mL) of FL46 complex peptide for 24 h showed no statistically significant difference in cell proliferation compared to the PBS group (P>0.05). This indicates that FL46 complex peptide at concentrations below 500 μg / mL does not inhibit the proliferation of U937 cells and has no toxic effect on them. Figure 5 ).

[0062] Example 3

[0063] Mouse immunization and Echinococcus granulosus protoscolex challenge infection

[0064] ① Six- to eight-week-old female C57BL / 6 mice were randomly divided into a peptide-French adjuvant group and a French adjuvant control group, with 10 mice in each group;

[0065] ② 50 μg of peptide vaccine FL46 (complex epitope peptide FL46) was emulsified and mixed with 50 μL of Freund's complete adjuvant (for primary immunization) or 50 μL of Freund's incomplete adjuvant (for the next two booster immunizations);

[0066] ③ C57BL / 6 mice were immunized three times by subcutaneous injection at multiple points on the back, with each injection spaced two weeks apart.

[0067] ④ Two weeks after each immunization, blood was collected from the orbital cavity of mice, and serum was separated to detect cytokine and antibody levels.

[0068] ⑤ Two weeks after the last immunization, mice in the polypeptide vaccine group and the control group were challenged by intraperitoneal injection of 2,000 Echinococcus granulosus protoscolex and housed in an SPF-grade animal facility for 8 months.

[0069] Example 4

[0070] MSD chip detection of serum cytokine expression levels in mice immunized with peptide-French adjuvant

[0071] ① The orbital blood sampling method involves collecting whole blood from mice 2 weeks after each immunization in Example 3, centrifuging at 3000g for 20 minutes at 4°C, and collecting the upper serum layer.

[0072] ② After adding 50 μL of 1×beads to each well of the 96-well plate, wash twice with washing buffer;

[0073] ③ Add 50 μL of standard, blank sample and serum sample to 96-well plate respectively, incubate on a shaker at 850 rpm for 1 h at room temperature, and wash 3 times with washing buffer;

[0074] ④ Add 25 μL of detection antibodies (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC / GRO, TNF-α) and incubate at room temperature on a shaker at 850 rpm for 30 min. Wash 3 times with washing buffer.

[0075] ⑤ Add 50 μL of 1×SA-PE and incubate on a shaker at 850 rpm at room temperature for 10 min, then wash 3 times;

[0076] ⑥ Add 125 μL of assay buffer and incubate at room temperature on a shaker at 850 rpm for 30 seconds;

[0077] ⑦ Place the 96-well plate on the Bio-Plex System for testing.

[0078] The results showed that mice immunized with the peptide-French adjuvanted vaccine had significantly higher serum IL-2 levels after a single immunization than mice immunized with Freund's adjuvant vaccine, and the expression level increased significantly with the number of immunizations. Two weeks after the last immunization with the peptide-French adjuvanted vaccine, the expression levels of TNF-α, IL-5, IL-10, and IL-12p70 in the serum of the vaccine-treated mice were significantly increased. Cellular immunity levels were also increased in mice immunized with the FL46 vaccine. Figure 7 )

[0079] Example 5

[0080] Indirect ELISA method for detecting the titers of IgM, IgG and their subtypes in the serum of mice after vaccine immunization.

[0081] ① Whole blood was collected from the orbital cavity of mice 2 weeks and 8 months after three immunizations with the polypeptide Freund's adjuvant in Example 3, as well as whole blood from mice immunized with Freund's adjuvant at the same time point in the control group. The cells were centrifuged at 3000 rpm for 15 min, and the supernatant serum was collected.

[0082] ② Dilute the antigen protein FL46 to 0.5 μg / ml with antigen coating buffer (pH 9.6) (absin), and add 50 μL to each well. Incubate overnight in a humidified chamber at 4°C. Discard the coating buffer, blot off any remaining coating buffer with filter paper, add 300 μL of PBST to each well, wash for 3-5 min, discard the PBST, and blot off any remaining PBST with filter paper. Repeat the washing process three times.

[0083] ③ Add 250 μL of blocking buffer (1% BSA) to each well. Block at 37°C for 2 hours; discard the blocking buffer in the wells, blot off any excess blocking buffer with filter paper, and wash the plate 3 times with PBST, following the same method as above;

[0084] ④ Primary antibody incubation: Dilute mouse serum 1:100 with PBST and add 100 μL of the diluted mouse serum to each well of the ELISA plate. Incubate the ELISA plate at 37°C for 1.5 hours (note that a negative control should be included). Discard the primary antibody serum from the wells, blot off any excess serum with filter paper, and wash the plate three times with PBST, following the same method as above.

[0085] ⑤ Secondary antibody incubation: Dilute goat anti-mouse IgM, IgG, IgG2a, and IgG1 labeled with horseradish peroxidase at a ratio of 1:10000 with PBST, and add 50 μL to each well. Incubate at 37°C for 1 hour, and wash the plate 3 times with PBST, following the same method as above.

[0086] ⑥ Color development: Add 50 μL of substrate solution (TMB) to each well and incubate at room temperature in the dark for 10 min; after color development, add 500 μL of stop solution to each well to terminate the reaction;

[0087] ⑦ Use an ELISA reader to read the absorbance at 450 nm.

[0088] The results showed that 32 weeks after the last immunization, the absorbance of total IgG and IgG2a in the serum of mice in the peptide-adjuvanted group was (0.42±0.16) and (0.19±0.15), respectively, which were significantly higher than those in the Freund's adjuvanted group (0.09±0.09 and (0.02±0.02). This indicates that immunization of mice with peptide-adjuvanted adjuvant induces humoral immunity, increases the immune level in mice, and antibody expression can persist for up to 8 months.

[0089] Example 6

[0090] Number, size, and weight of echinococcosis larvae in mice immunized with the vaccine after being challenged with granular echinococcosis larvae

[0091] ① Mice infected with Echinococcus granulosus larvae for 8 months after the last immunization with the polypeptide Freund's adjuvant and Freund's adjuvant in Example 3 were anesthetized by intraperitoneal injection of 0.1% sodium pentobarbital and euthanized by cervical dislocation.

[0092] ② The abdominal cavity was opened, and the size and number of cysts in the abdominal cavity of the two groups of mice were observed with the naked eye;

[0093] ③ Gently remove all cysts, weigh each mouse cyst, and record its weight.

[0094] The results showed that compared with mice immunized with Freund's adjuvant, the size and number of cysts in the peptide Freund's adjuvant group were significantly reduced, and the cyst weight was significantly reduced by (59.16±1.83)%. Figures 10-11 This indicates that the FL46 peptide vaccine has a protective effect against Echinococcus granulosus infection and can reduce the infection level of Echinococcus granulosus.

[0095] In summary, this invention utilizes linkers to connect multiple antigenic epitopes, successfully constructing the complex polypeptide FL46 and enhancing its immunogenicity. Furthermore, this invention co-constitutes FL46 with Freund's adjuvant to create the FL46 polypeptide Freund's adjuvant vaccine, and verifies its protective effect against mouse echinococcosis, achieving significant cyst reduction. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and possesses high industrial applicability.

[0096] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A complex antigen polypeptide, characterized in that, The amino acid sequence of the complex antigen polypeptide is shown in SEQ ID NO:

4.

2. A vaccine for echinococcosis, characterized in that, The echinococcosis vaccine comprises the complex antigen polypeptide as described in claim 1.

3. The echinococcosis vaccine according to claim 2, characterized in that, The echinococcosis vaccine is a Echinococcus granulosus vaccine.

4. The echinococcosis vaccine according to claim 3, characterized in that, The Echinococcus granulosus vaccine comprises: The complex antigen polypeptide of claim 1; and Freund's complete adjuvant or Freund's incomplete adjuvant.

Images