Multi-antigen fusion protein based on sprm1hc extracellular region and preparation method and application thereof

Abstract

The application provides a multi-antigen fusion protein taking an SPRM1hc extracellular region as a main body, a preparation method and application thereof, and belongs to the technical field of parasite immunology, vaccine engineering and biopharmaceutical technology. The application first proposes that the SPRM1hc extracellular region (ECD) is used as a vaccine antigen, and the extracellular region of the SPRM1hc is fused with the antigen epitopes of the classic protective antigens Sj23 and SjTSP2 to construct a new multi-antigen fusion protein. The immunization effect is significantly enhanced through a combined immunization strategy. A vaccine composition is prepared based on the fusion antigen, and the effective application of the vaccine composition in the prevention of schistosome infection is verified.

Description

Technical Field

[0001] This invention belongs to the fields of parasitic immunology, vaccine engineering, and biopharmaceutical technology. Specifically, it relates to a multi-antigen fusion protein constructed by combining the extracellular region (ECD) of the transmembrane protein SPRM1hc of Schistosoma japonicum as the core antigen with the epitope regions of protective antigens Sj23 and SjTSP2, as well as a vaccine composition prepared based on this fusion antigen and its application in the prevention of schistosomiasis infection. Background Technology

[0002] Schistosomiasis is a major parasitic disease in tropical and subtropical regions, posing a serious threat to human health. Current treatment primarily relies on chemical drugs, such as praziquantel; however, these drugs do not provide durable immune protection, and long-term use may lead to drug resistance. Therefore, developing vaccines that can induce potent humoral and cellular immunity is crucial for controlling schistosomiasis.

[0003] Existing anti-schistosomiasis vaccines mostly focus on surface antigens such as Sj23 and SjTSP2. While these transmembrane proteins can elicit a certain immune response, their protective effect has not yet reached an ideal level. SPRM1hc protein, a transmembrane protein of Schistosoma japonicum, has its extracellular domain (ECD) exposed on the surface of the parasite, making it a potential vaccine candidate antigen. Although studies have shown that this protein plays a role in the immune response, there are no reports of SPRM1hc ECD being used as an independent vaccine antigen. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a multi-antigen fusion protein based on the extracellular region of SPRM1hc, its preparation method, and its applications. This invention is the first to propose using the extracellular region (ECD) of SPRM1hc as a vaccine antigen, and to fuse the extracellular region of SPRM1hc with the epitopes of the classic protective antigens Sj23 and SjTSP2 to construct a novel multi-antigen fusion protein. This protein significantly enhances the immune effect through a combined immunization strategy, thereby providing an effective schistosomiasis vaccine.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an ECD antigen of the transmembrane protein SPRM1hc from Schistosoma japonicum, wherein the antigen is the natural extracellular region from position 90 to the C-terminus of the full-length sequence of the SPRM1hc protein, and its amino acid sequence is shown in SEQ ID NO:1.

[0006] Secondly, the present invention provides a multi-antigen fusion protein antigen with the extracellular region of SPRM1hc as the main body. It is composed of the extracellular region of PRM1hc with the antigenic region of Sj23 or / and SjTSP2, as shown in SEQ ID NO:1, including SPRM1hc-Sj23 fusion antigen, SPRM1hc-SjTSP2 fusion antigen and SPRM1hc-Sj23-SjTSP2 triple fusion antigen.

[0007] As a further optimization of the above-mentioned multi-antigen fusion protein antigens, the amino acid sequence of the SPRM1hc-Sj23 fusion antigen is shown in SEQ ID NO:3; the amino acid sequence of the SPRM1hc-SjTSP2 fusion antigen is shown in SEQ ID NO:5; and the amino acid sequence of the SPRM1hc-Sj23-SjTSP2 triple fusion antigen is shown in SEQ ID NO:7.

[0008] Thirdly, the present invention provides a method for preparing the above-mentioned antigen, comprising the following steps: (1) The antigen gene was artificially synthesized and amplified to form the target fragment by PCR splicing according to different antigen combination forms; (2) The obtained target fragment is digested with two enzymes and then ligated with an expression vector suitable for transient expression in eukaryotic cells to construct a recombinant expression plasmid; (3) The recombinant plasmid was transiently transfected into HEK293F cells and expressed under conventional suspension culture conditions; (4) After expression, the culture supernatant was centrifuged, clarified and filtered before entering the purification step. First, Ni-NTA coupling affinity chromatography was used for preliminary purification, and then molecular sieve chromatography was used for further purification to obtain the purified product for storage.

[0009] Fourthly, the present invention provides the use of the above-mentioned antigen in the preparation of a vaccine formulation against Schistosoma japonicum. Further, the vaccine formulation is a recombinant protein vaccine, a DNA vaccine, an mRNA vaccine, or other known forms of immune delivery in the art (such as mosquito delivery vectors).

[0010] Fifthly, the present invention provides a vaccine composition for combating Schistosoma japonicum infection, comprising the antigen described above.

[0011] As a further optimization of the above-mentioned vaccine composition, it is prepared by mixing the antigen with Freund's adjuvant in a certain proportion. Even further, it is prepared by mixing the antigen with Freund's adjuvant containing CpG2 in a certain proportion.

[0012] In a sixth aspect, the present invention provides the use of the above-described vaccine composition in the preparation of a medicament against Schistosoma japonicum infection.

[0013] Beneficial effects: 1. This invention is the first to use SPRM1hc ECD as a vaccine antigen, which is novel. This antigen has not been previously reported in the literature as a vaccine target, demonstrating strong innovation.

[0014] 2. The multi-antigen fusion protein strategy with the extracellular region of SPRM1hc as the main component can significantly enhance immunogenicity. In particular, the SPRM1hc-Sj23-SjTSP2 triple fusion antigen can induce higher antibody titers, immune responses mainly enhanced by Th1, and stronger TNF-α responses.

[0015] 3. SPRM1hc, SPRM1hc-Sj23, SPRM1hc-SjTSP2 and the triple fusion antigen SPRM1hc-Sj23-SjTSP2 of the present invention all have significant protective effects against infection, manifested as: significantly reducing the number of adult worms, significantly reducing the egg load, and significantly alleviating liver damage and fibrosis.

[0016] 4. The fusion antigen platform can be extended to various vaccine forms, including recombinant protein vaccines, DNA vaccines, mRNA vaccines, and mosquito delivery vectors known in the art.

[0017] 5. The design strategy of this invention is universal, and the multi-antigen fusion approach can be extended to the development of other parasite vaccines. Attached Figure Description

[0018] Figure 1 The sequence maps are for SPRM1hc-ECD, SPRM1hc-Sj23, SPRM1hc-SjTSP2, and SPRM1hc-Sj23-SjTSP2; among them, the SPRM1hc-ECD protein has an N-terminal His tag, and the other fusion antigens have a C-terminal His tag.

[0019] Figure 2 The images show molecular sieve spectra and SDS-PAGE results for SPRM1hc-ECD, SPRM1hc-Sj23, SPRM1hc-SjTSP2, and SPRM1hc-Sj23-SjTSP2.

[0020] Figure 3 The graph shows the results of the assessment of the number of adult worms recovered and the worm reduction rate after immunization.

[0021] Figure 4 This is a graph showing the assessment results of the parasite egg load and egg reduction rate in liver tissue after immunization.

[0022] Figure 5 The graph shows the changes in serum IgG antibody levels in mice after immunization with SPRM1hc and its fusion antigen.

[0023] Figure 6 The graph shows the changes in serum IgG2A antibody and the IgG2A / IgG1 ratio in mice after immunization.

[0024] Figure 7 This is a graph showing the changes in IgG1, IgG2B, Ig3, and IgM levels after immunization.

[0025] Figure 8 The figure shows the results of Th1 cell immune responses induced by various antigens. In the figure, the cellular immune response is mainly enhanced by Th1: the expression levels of IL-2 and TNF-α in the spleen cells of the immune group are higher than those in the control group, and the expression level of IFN-γ in the spleen cells of the SPRM1hc+SjTSP2 group is higher than that in the control group.

[0026] Figure 9 The figure shows the results of Th1 cell immune response after the addition of CpG2; in the figure, CpG2 + Freund's adjuvant enhanced the Th1 cell immune response: the expression level of TNF-α in the spleen cells of the immunization group using CpG2 + Freund's adjuvant was slightly higher than that in the control group and the Freund's adjuvant alone group; IFN-γ: interferon-γ; IL-2: interleukin-2; TNF-α: α tumor necrosis factor.

[0027] Figure 10 The figure shows the results of Th2 cell immune responses induced by various antigens; in the figure, the overall Th2 cell immune response after immunization showed a downward trend; IL-4: interleukin-4; IL-10: interleukin-10; IL-6: interleukin-6; IL-13: interleukin-13.

[0028] Figure 11 The image shows the pathological analysis results of liver tissue after SRRM1hc immunization; in the image, HE: hematoxylin-eosin staining; Masson: Masson trichrome staining.

[0029] Figure 12 The image shows the pathological analysis results of liver tissue after SPRM1hc combined with antigen immunization; in the image, HE: hematoxylin-eosin staining; Masson: Masson trichrome staining.

[0030] above Figures 3-4 , Figure 8-10 Medium, ns: P ≥ 0.05; *: 0.01 ≤ P < 0.05; **: 0.001 ≤ P < 0.01; ***: P < 0.001; ****: P < 0.0001. Detailed Implementation

[0031] The present invention aims to provide a novel multi-antigen fusion protein antigen composed of the extracellular region (ECD, 90–end) of SPRM1hc and the antigenic regions of Sj23 and SjTSP2, and to prepare a vaccine composition with significant immunoprotective effect based on this antigen.

[0032] I. Construction of Multi-Antigen Fusion Proteins 1. Core structure: SPRM1hc ECD (hc 90–end).

[0033] This invention identifies the amino acid sequence from position 90 to the C-terminus of the full-length SPRM1hc protein as its natural extracellular region (ECD). Its characteristics include: protein structure exposed to the host environment, easy recognition by B cells and APCs, and the presence of multiple antigenic epitopes, making it an ideal candidate antigen for vaccines. Through gene amplification (polymerase chain reaction), restriction endonuclease digestion (KpnⅠ and XhoⅠ), ligation (T4 ligase), positive clone screening, plasmid extraction, and sequencing, a recombinant protein expression vector for the SPRM1hc protein extracellular region was finally obtained, with a His tag at its N-terminus.

[0034] The amino acid and nucleotide sequences of SPRM1hc-ECD are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0035] 2. The fusion antigen constructed in this invention further includes: (1) SPRM1hc-Sj23 fusion antigen: SPRM1hc is connected to the Sj23 antigen region with ECD as the N end.

[0036] (2) SPRM1hc-SjTSP2 fusion antigen: SPRM1hc has ECD as the N-terminus; connected to the immune-related domain of SjTSP-2.

[0037] (3) SPRM1hc-Sj23-SjTSP2 triple fusion antigen (the focus of this invention): SPRM1hc ECD; Sj23 antigen domain; SjTSP2 antigen domain; linearly fused in sequence or linked by a flexible linker. This triple fusion antigen has been proven to have the best immunogenic effect and is the core object of protection of the claims.

[0038] The amino acid and nucleotide sequences of the SPRM1hc-Sj23 fusion antigen are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0039] The amino acid and nucleotide sequences of the SPRM1hc-SjTSP2 fusion antigen are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0040] The amino acid and nucleotide sequences of the SPRM1hc-Sj23-SjTSP2 triple fusion antigen are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0041] All fusion proteins can be tagged with His-tags, and the protection scope includes any tag variant. Furthermore, although the extracellular region (ECD) of SPRM1hc is used as a preferred embodiment in constructing the fusion protein, it should be understood that this provides broader protection for the application of SPRM1hc in vaccines. Full-length SPRM1hc protein, truncated fragments, homologous variants, or equivalent sequences capable of replacing the ECD fragment to perform immune-inducing functions are also within the protection scope of this invention.

[0042] II. Expression and Purification of Fusion Proteins The recombinant plasmid was used for transient transfection of HEK293F cells and expressed for approximately 72 hours under logarithmic growth phase suspension culture conditions at 37°C. The expression supernatant was clarified and then preliminarily purified by (1) Ni-NTA-coupled affinity chromatography and (2) molecular sieve chromatography. The purified product was stored in PBS for subsequent immunoassays.

[0043] III. Vaccine Formulation The adjuvant systems include: complete / incomplete Freund's adjuvants; and CpG2 ODN (a novel adjuvant).

[0044] Antigen dosage (12 mice per group): 1200 μg protein (100 μg / mouse); 60 μg CpG2 ODN (5 μg / mouse).

[0045] Antigen:Adjuvant = 1:1 (volume ratio).

[0046] IV. Mouse Immunization Procedure Immunization was administered in weeks 0, 2, and 4; blood was collected every 2 weeks for antibody testing; spleen cells were collected on day 14 of immunization for cellular immunoassay.

[0047] V. Immune Response Detection (1) ELISA detection of specific antibody subtypes IgG, IgG1, IgG2a, IgG2b, IgG3, IgM.

[0048] Used to assess total antibody response and Th1 / Th2 bias.

[0049] (2) Cytokine detection A. Flow cytometry (intracellular) Th1: IFN-γ, IL-2, TNF-α Th2: IL-4, IL-6, IL-10, IL-13 B. Spleen cell in vitro stimulation supernatant (ELISA) Stimulus conditions: 6 × 10 5 / well; protein concentration 10 µg / mL; culture for 72 h (37°C, 5% CO2).

[0050] Cytokines detected: IL-2, IL-4, IL-10, IFN-γ, IL-6, TNF-α.

[0051] VI. Evaluation of Infection and Protective Effects Infection conditions: Each mouse was infected with 35±1 Schistosoma japonicum cercariae; the mice were sacrificed 6 weeks after infection.

[0052] Evaluation indicators: (1) Number of adult worms recovered by portal vein perfusion; (2) Body length of male and female worms; (3) Number of worm eggs in liver tissue (digested with 5% KOH); (4) Worm reduction rate and egg reduction rate; (5) Fecal worm egg count; (6) Liver tissue pathological section (degree of fibrosis).

[0053] The triple fusion antigen SPRM1hc-Sj23-SjTSP2 group exhibited significant immunoprotective effects, which is one of the core innovations of this invention.

[0054] The following uses SPRM1hc-ECD as an exemplary antigen to illustrate the construction and purification process of this invention. However, those skilled in the art will understand that the full-length sequence, functionally equivalent fragments, or homologous variants of SPRM1hc can be used to replace SPRM1hc-ECD and are still within the scope of this invention.

[0055] Unless otherwise specified, the reagents or materials used in the following examples are all commercially available, and the operations used are all conventional technical means.

[0056] Example 1: Construction and expression of antigen.

[0057] (1) The coding sequences of SPRM1hc, SPRM1hc-Sj23, SPRM1hc-SjTSP2 and the triple fusion antigen SPRM1hc-Sj23-SjTSP2 of the present invention were all artificially synthesized by a commercial gene synthesis company according to the reference sequence of the corresponding gene of Schistosoma japonicum. Subsequently, according to different antigen combination forms, the required fusion gene fragments were amplified by PCR splicing method.

[0058] The obtained target fragment was double-digested with KpnI / XhoI and then ligated into an expression vector suitable for transient expression in eukaryotic cells to construct a recombinant expression plasmid. During construction, the SPRM1hc-ECD protein carried an N-terminal His tag, while the other fusion antigens carried C-terminal His tags. Figure 1 This facilitates a standardized affinity purification process. After sequencing verification, the recombinant plasmid was used for transient transfection of HEK293F cells.

[0059] (2) 293F cells were expressed for approximately 72 hours under standard suspension culture conditions (37℃, 5% CO2, 130 rpm). After expression, the culture supernatant was clarified by centrifugation and filtered before proceeding to the purification step. Initial purification was performed first using Ni-NTA affinity chromatography. Subsequently, the Ni elution product was further purified by molecular sieve chromatography to remove aggregates and obtain a more homogeneous protein fraction. Figure 2 The molecular sieve conditions used in this invention can effectively improve protein homogeneity, ultimately yielding antigen proteins with high purity, sufficient to meet the needs of subsequent immunoassays.

[0060] (3) The purified protein is stored in PBS-based buffer and can be cryopreserved for a long time or used for short-term animal immunization, in vitro stimulation experiments, and related immunological detection. The expression and purification procedures described in this embodiment are routine methods that can be repeatedly implemented by those skilled in the art.

[0061] Example 2: Preparation of vaccine formulation.

[0062] The purified fusion antigen was mixed with Freund's adjuvant at a 1:1 volume ratio. hc(90-end) was then mixed with either Freund's adjuvant alone or with Freund's adjuvant containing CpG2 at a 1:1 volume ratio to prepare a liquid vaccine formulation. This formulation is suitable for mouse immunization experiments.

[0063] Example 3: Immunoprotection experiment.

[0064] Mice were vaccinated three times (at weeks 0, 2, and 4) according to the immunization protocol, followed by a challenge infection experiment. Six weeks post-infection, the effectiveness of the immunoprotective effect was assessed, including adult worm recovery rate, egg load, and pathological analysis.

[0065] Example 4: Evaluation of adult worm recovery and reduction rate after immunization ( Figure 3 ).

[0066] Mice were challenged with cercariae after immunization, and adult worms were recovered via portal vein perfusion at week 6 after infection.

[0067] The average number of adults and the reduction rate for each group are as follows:

[0068] Example 5: Assessment of egg load and egg reduction rate in liver tissue ( Figure 4 ).

[0069] After the experiment, the liver was dissected, weighed, and digested with KOH. The number of parasite eggs per unit liver tissue was calculated.

[0070] The results are as follows:

[0071] Example 6: Analysis of dynamic changes in antibody titers after immunization.

[0072] To investigate the types and expression of antibodies in mice after immunization, mice were infected 6 weeks after immunization, and serum was collected at weeks 0, 2, 4, and 6. The titer of antigen-specific IgG antibodies was detected by ELISA.

[0073] The results show that: 1. All SPRM1hc-related antigens can significantly increase IgG titers ( Figure 5 ).

[0074] 2. IgG2A and the IgG2A / IgG1 ratio significantly increased after immunization and remained at high levels for a prolonged period after infection, suggesting that they may be the antibodies playing a major role in immune protection. Meanwhile, IgG2A is the main antibody in the Th1 immune response; therefore, the Th1 immune response is the primary agent of immune protection. Figure 6 ).

[0075] 3. IgG1, IgG2B, Ig3, and IgM all increase after immunization, but cannot maintain high levels after infection. Figure 7 ).

[0076] The above results indicate that SPRM1hc itself has good B cell immunogenicity, and the multi-antigen fusion strategy, especially the triple fusion antigen, can significantly improve antibody levels; the core antibody for immune protection is IgG2A, which depends on the Th1 immune response.

[0077] Example 7: Flow cytometry analysis of Th1 / Th2 cell immune response.

[0078] To further evaluate the cellular immune responses induced by each antigen, mouse spleen cells were harvested on day 14 after the last immunization and stimulated with PMA / Ionomycin for flow cytometry analysis of intracellular cytokines (IFN-γ, IL-2, TNF-α, IL-4, IL-10, etc.).

[0079] The results show that: Th1 response: SPRM1hc and its fusion antigen can both induce a Th1 response: IL-2 and TNF-α are upregulated. Figure 8 The addition of CpG2 can enhance the expression of IL-2 and TNF-α. Figure 9 ).

[0080] Th2 response: The expression levels of IL-4, IL-10, IL-6, and IL-13 in the spleen cells of the immunized group showed a decreasing trend compared with the control group. Figure 10 ); The above results indicate that the fusion antigen of the present invention dominates the Th1-type response and can activate a significant cellular immune response.

[0081] Example 8: Liver tissue pathological analysis (HE and Masson staining) Figure 11 () Figure 12 ).

[0082] To verify the vaccine's effect on improving liver damage after infection, liver tissue was taken 6 weeks after the challenge infection for HE staining and Masson staining.

[0083] The main results are as follows: HE staining: Extensive inflammatory cell infiltration and granuloma formation were observed in the PBS and FA groups; the degree of inflammation was reduced in the SPRM1hc, SPRM1hc+Sj23, and SPRM1hc+SjTSP2 groups; the triple fusion protein group showed the mildest inflammatory response and a significant reduction in granuloma area.

[0084] Masson staining: A large amount of collagen fiber deposition was observed in the control group (PBS, FA); the degree of fibrosis was improved in the single antigen group; the fibrosis was significantly reduced in the triple fusion group, which was the best among all groups.

[0085] This indicates that the triple fusion antigen not only reduces the burden of parasites and eggs, but also significantly improves the pathological damage to liver tissue caused by schistosomiasis infection.

Claims

1. A Schistosoma japonicum vaccine antigen, characterized in that: The vaccine antigen is a transmembrane protein SPRM1hc ECD antigen, characterized in that: the antigen is the natural extracellular region from position 90 to the C-terminus of the full-length sequence of the SPRM1hc protein, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A multi-antigen fusion protein antigen with the SPRM1hc extracellular region as the main component, characterized in that: It is constructed by fusing the extracellular region of SPRM1hc, as shown in SEQ ID NO:1, with the antigenic region of Sj23 or / and SjTSP2, including SPRM1hc-Sj23 fusion antigen, SPRM1hc-SjTSP2 fusion antigen and SPRM1hc-Sj23-SjTSP2 triple fusion antigen. The amino acid sequence of the SPRM1hc-Sj23 fusion antigen is shown in SEQ ID NO:3; The amino acid sequence of the SPRM1hc-SjTSP2 fusion antigen is shown in SEQ ID NO:5; The amino acid sequence of the SPRM1hc-Sj23-SjTSP2 triple fusion antigen is shown in SEQ ID NO:

7.

3. The method for preparing the antigen according to claim 1 or 2, characterized in that: Includes the following steps: (1) The antigen gene was artificially synthesized and amplified to form the target fragment by PCR splicing according to different antigen combination forms; (2) The obtained target fragment is digested with two enzymes and then ligated with an expression vector suitable for transient expression in eukaryotic cells to construct a recombinant expression plasmid; (3) The recombinant plasmid was transiently transfected into HEK293F cells and expressed under conventional suspension culture conditions; (4) After expression, the culture supernatant is centrifuged, clarified and filtered before entering the purification step. The purification step is to first perform preliminary purification using Ni-NTA coupling affinity chromatography, and then further purify using molecular sieve chromatography to obtain the purified product for storage.

4. The use of the antigen according to claim 1 or 2 in the preparation of a vaccine formulation against Schistosoma japonicum.

5. The application according to claim 4, characterized in that: The vaccine formulation is a recombinant protein vaccine, a DNA vaccine, an mRNA vaccine, or a mosquito delivery vector.

6. A vaccine composition against Schistosoma japonicum infection, characterized in that: Includes the antigen described in claim 1 or 2.

7. The vaccine composition according to claim 6, characterized in that: It is prepared by mixing the antigen with Freund's adjuvant in a certain proportion.

8. The vaccine composition according to claim 7, characterized in that: It is prepared by mixing the antigen with Freund's adjuvant containing CpG2 in a certain proportion.

9. The use of the vaccine composition according to claim 6 in the preparation of a medicament against Schistosoma japonicum infection.

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