Multi-epitope fusion antigen vaccine derived from plasmodium falciparum STEVOR protein as well as preparation and application of multi-epitope fusion antigen vaccine

Abstract

The invention relates to the field of molecular vaccinology, in particular to a vaccine aiming at severe malaria, and further relates to a multi-epitope fusion antigen vaccine derived from plasmodium falciparum STEVOR protein as well as preparation and application of the multi-epitope fusion antigen vaccine. The multi-epitope fusion antigen vaccine contains at least two amino acid fragments in a B cell epitope, a CD4 and T cell epitope and a CD8 and T cell epitope. The MEFA vaccine targeting STEVOR protein SC structural domain conservative immunodominant epitopes has broad spectrum, can realize 97.15% global HLA coverage rate through the conservative epitopes, can synchronously induce IgG antibody and CD4 + / CD8 + T cell response, is safe, has no toxicity / sensitization, and has good in-vivo and in-vitro stability (the mammalian half-life period gt; further, the strain is used for inducing wide immune response for resisting severe malaria, and is suitable for large-scale production of an escherichia coli or yeast expression system.

Description

Technical Field

[0001] The present invention relates to the field of molecular vaccinology, in particular to vaccines against severe malaria, and more particularly to a multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum and its preparation and application. Background Art

[0002] Malaria is one of the most serious infectious diseases worldwide, with Plasmodium falciparum causing the majority of severe cases and deaths. According to the World Health Organization (WHO), approximately 249 million cases of malaria and 609,000 deaths were reported worldwide in 2022, primarily affecting children under five and pregnant women in sub-Saharan Africa. Over 200 species of Plasmodium have been identified, but only five routinely infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Of these five, Plasmodium falciparum is the leading cause of malaria-related deaths due to the severity of the disease and numerous complications it causes. Despite decades of research, no vaccine currently fully prevents severe malaria. Existing interventions, such as insecticide-treated mosquito nets and artemisinin combination therapy, face challenges with insecticide and parasite resistance. Therefore, the development of vaccines targeting conserved immunogenic regions of P. falciparum is crucial to reduce morbidity and mortality in endemic areas.

[0003] The current mainstream malaria vaccine RTS,S / AS01 (Mosquirix) targets the circumsporozoite protein (CSP), but its protective efficacy is limited (about 30-40%) and weakens over time. Other candidate vaccines (such as the VAR2CSA vaccine based on the PfEMP1 protein family) only target specific variants (such as placental malaria) and cannot cope with the wide antigenic diversity of Plasmodium falciparum. This diversity stems from the polymorphic variant surface antigens (VSAs, including STEVORs, RIFINs and PfEMP1s) expressed by the parasite. These antigens aggravate pathological damage and cause severe malaria through immune escape and red blood cell rosette formation. The core difficulty in vaccine design lies in how to identify universal protective epitopes from highly variable antigens. There is an urgent need to develop innovative strategies targeting conserved functional regions to induce broad and lasting immunity.

[0004] STEVORs (subtelomeric variable open reading frames) are a family of proteins expressed on the surface of Plasmodium falciparum-infected erythrocytes (iRBCs) and are involved in the pathogenesis of severe malaria. They regulate erythrocyte deformability by mediating rosetting (iRBCs binding to uninfected erythrocytes, obstructing microvascular blood flow) and interacting with glycophorin C (GPC). Unlike other VSAs, the SC domain of STEVORs is exposed on the iRBC surface and recognized by antibodies from individuals exposed to malaria. Recent studies have shown that anti-STEVOR antibodies can inhibit rosetting, suggesting that immune responses targeting these proteins have a protective effect. However, the specific epitopes in STEVOR that drive protective immunity have not been identified, hindering rational vaccine design.

[0005] Traditional malaria vaccine strategies are limited by issues such as antigenic diversity, low immunogenicity, and delivery efficiency. Whole-protein vaccines often contain variants or immunosuppressive regions, while epitope vaccines provide precise solutions by focusing on conserved immunodominant sequences. However, how to screen B / T cell epitopes that are both highly conserved and strongly immunogenic remains a challenge. The development of computational immunology has provided a powerful tool for predicting high-affinity epitopes of MHC molecules and broad population coverage, but there are challenges in simulating the complexity of immune responses or in the evolutionary adaptability of pathogens. At the same time, converting computer predictions into clinically available multi-epitope vaccines still has scientific uncertainties at the basic prediction level (such as errors in epitope immunogenicity predictions, blind spots in immune response regulation), or specific risks in the clinical stage (such as challenges of population heterogeneity, defects in immune persistence, and risks of immune enhancement), which require further exploration. Summary of the Invention

[0006] The present invention aims to provide a multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum and its preparation and application.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] A multi-epitope fusion antigen vaccine derived from the STEVOR protein of Plasmodium falciparum, which contains at least two amino acid fragments of B cell epitopes, CD4 and T cell epitopes, and CD8 and T cell epitopes.

[0009] The multi-epitope fusion antigen vaccine contains at least two of the amino acid sequences described in SEQ ID No: 1-29.

[0010] The multi-epitope fusion antigen vaccine is the amino acid sequence of SEQ ID No: 1-29 and is connected by a connecting peptide.

[0011] The multi-epitope fusion antigen vaccine is the amino acid sequence of SEQ ID No: 1-29 and is connected to the β-defensin-3 adjuvant via a connecting peptide; wherein the β-defensin-3 adjuvant amino acid sequence is shown in SEQ ID No: 30.

[0012] The amino acid sequences of the CD8 and T cell epitopes are SEQ ID Nos: 1-9; the amino acid sequences of the CD4 and T cell epitopes are SEQ ID Nos: 10-20; and the amino acid sequences of the B cell epitopes are SEQ ID Nos: 21-29.

[0013] The multi-epitope fusion antigen vaccine is composed of an adjuvant (β-defensin-3), a connecting peptide (EAAAK), CD8 and T cell epitope amino acid sequences, a connecting peptide (GPGPG), CD4 and T cell epitope amino acid sequences, a connecting peptide (KK), and a B cell epitope amino acid sequence.

[0014] A method for constructing a multi-epitope fusion antigen vaccine derived from the STEVOR protein of Plasmodium falciparum comprises connecting an adjuvant to a CD8+ T cell epitope (SEQ ID No. 1-9) using a rigid connecting peptide, and then bridging the CD8+ T cell epitope with the CD4+ T cell epitope (SEQ ID No. 10-20) via a "GPGPG" connecting peptide; the B cell epitope (SEQ ID No. 21-29) is connected to the CD4+ T cell epitope via a connecting peptide (KK), and then the sequences in the B cell epitope are interconnected via the KK connecting peptide.

[0015] The sequences in the CD8+T cell epitopes (SEQ ID No. 1-9) are interconnected through an AAY connecting peptide; the sequences in the CD4+T cell epitopes (SEQ ID No. 10-20) are interconnected through a GPGPG connecting peptide; and the sequences in the B cell epitopes (SEQ ID No. 11-29) are interconnected through a KK connecting peptide.

[0016] An application of the multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum, and the application of the vaccine as a malaria vaccine.

[0017] The advantages of the present invention are:

[0018] This study identified and validated conserved multiple epitopes within the STEVORs SC domain through a combined computational and experimental approach. The MEFA construct, designed based on high antigenicity, low entropy, and broad HLA binding properties, overcomes the limitations of antigenic diversity while synergistically stimulating humoral and cellular immunity. This strategy leverages the critical role of the STEVORs SC domain in innate immunity and draws on the successful experience of multi-epitope vaccines in other infectious diseases. The introduction of the β-defensin-3 adjuvant further enhanced immunogenicity, providing an innovative solution for malaria vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram illustrating the identification of B cell and T cell epitopes in the STEVOR protein provided in an embodiment of the present invention; wherein A is PF3D7_1040200 (ST1), B is PF3D7_0617600 (ST2), C is PF3D7_0115400 (ST3), and D is PF3D7_0300400 (ST4).

[0020] Figure 2 This is a diagram showing the difference in average amino acid mutation rates of multiple epitopes provided in an embodiment of the present invention.

[0021] Figure 3 This is a diagram showing the effect of specific IgG antibodies against epitope peptides in mice immunized with the multi-epitope peptides provided in an embodiment of the present invention.

[0022] Figure 4 This is a diagram showing the effects of IL-2 and IFN-γ levels in mice immunized with the multi-epitope peptides provided in an embodiment of the present invention.

[0023] Figure 5 This is a comparative effect diagram of the number of T cells in mice immunized with the multi-epitope peptide segments provided in the examples of the present invention.

[0024] Figure 6 This is the design of the MEFA vaccine construct provided in the embodiments of the present invention.

[0025] Figure 7 A structural diagram of the MEFA construct provided in an embodiment of the present invention; wherein A is a three-dimensional structural model diagram, and B is a Ramachandran diagram.

[0026] Figure 8Figure 1 is a diagram showing the recombinant expression and purification effects of the META vaccine construct provided in an embodiment of the present invention, wherein A is SDS-PAGE analysis (lane M1: protein marker, Bio-Rad, product number 1610374S, the size of the marker band refers to the template on the left), and B is Western blot analysis (lane M2: protein marker, GenScript, product number M00673, the size of the marker band refers to the template on the left).

[0027] BSA: 2.00 μg).

[0028] Figure 9 Figures A, B, and C show that the dose simulations all stimulated significant T cell immune responses, while Figures D, E, and F show B cell immune responses. DETAILED DESCRIPTION

[0029] The present invention is described below by way of examples. It should be noted that the specific embodiments described herein are only intended to illustrate and explain the present invention, and are not intended to limit the present invention.

[0030] The present invention focuses on the semi-conservative functional domains of four kinds of STEVOR proteins (PF3D7_1040200, PF3D7_0617600, PF3D7_0115400 and PF3D7_0300400, hereinafter referred to as ST1-ST4).By computational immunology methods, relatively conservative, with high immunogenicity, no toxic side effect, no sensitization and hydrophilic T / B cell multi-epitopes are obtained, and the conservation of these multi-epitopes is verified based on homologous isolate sequences, and then it is combined into fusion multi-epitope units.Then the antigenicity and safety immunogenicity of the fusion multi-epitopes are further assessed (that is, anti-STEVOR IgG antibody levels are detected in clinical serum samples and immune mice of Plasmodium falciparum infected patients, and the correlation between the conservation of multi-epitope amino acids and antibody response is analyzed simultaneously), and then multi-epitope fusion antigen (MEVA) vaccine constructs are obtained, and its immunogenicity is verified by computer simulation.

[0031] The MEFA vaccine of the present invention targets the conserved immunodominant epitopes of the SC domain of the STEVOR protein. It has a broad spectrum and achieves 97.15% global HLA coverage through conserved epitopes. It also has the ability to simultaneously induce IgG antibodies and CD4+ / CD8+ T cell responses (VaxiJen antigenicity: 0.6855). It is safe and non-toxic / allergenic, and has good in vitro and in vivo stability and hydrophilicity (-1.169, 22.61; mammalian half-life >30 hours). It is a multi-epitope subunit candidate vaccine for inducing a broad immune response against severe malaria and is suitable for large-scale production in Escherichia coli or yeast expression systems.

[0032] Example 1

[0033] Epitope screening

[0034] Protein sequence acquisition and domain localization: The STEVOR protein sequences of Plasmodium falciparum 3D7 strain (ST1: PF3D7_1040200; ST2: PF3D7_0617600; ST3: PF3D7_0115400; ST4: PF3D7_0300400) were obtained from the PlasmoDB database, and multiple sequence alignment was performed using Jalview software (https: / / www.jalview.org / ). Signal peptide prediction was then performed using SignalP-6.0 (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ), and transmembrane domain localization analysis was completed in combination with DeepTMHMM (https: / / dtu.biolib.com / DeepTMHMM).

[0035] Computational prediction of B-cell and T-cell epitopes: Based on the transmembrane domain located above, linear B-cell epitopes in the SC domain were predicted using the B-cell epitope prediction tool of the Immune Epitope Database (IEDB) (http: / / tools.iedb.org / main / bcell / ), and epitopes with a threshold >0.55 were screened. At the same time, the three-dimensional structural models of the four STEVOR proteins were analyzed using ElliPro (http: / / tools.iedb.org / ellipro / ) to verify discontinuous epitopes. For T cell epitopes: IEDB Next Generation Tools (https: / / nextgen-tools.iedb.org / pipeline) were used to screen for strong MHC-I binding CD8+ T cell epitopes with a percentile value <0.5 as a threshold. The IEDB MHC-II Binding Simulation System (http: / / tools.iedb.org / mhcii / ) was used to screen for CD4+ T cell epitopes with a percentile value <2. All T cell epitope identification was based on a complete human leukocyte antigen (HLA) reference dataset. That is, the IEDB and VaxiJen computational tools were used to predict B cell and T cell epitopes in the ST1-ST4 sequences obtained above. The screening criteria were: antigenicity score >0.5, entropy value <0.5, and HLA binding affinity (MHC-I percentile rank <0.5, MHC-II <2).

[0036] Analysis of epitope physicochemical properties: The B cell epitopes obtained above that overlapped with CD8+ and CD4+ T cell epitopes were selected as polytope candidates, and their low complexity regions were analyzed using the PlasmoDB database. The hydrophilicity, surface accessibility, and antigenicity of the epitopes were then comprehensively evaluated using the DNAStar Protean system and the Vaxijen online platform (https: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.html). Finally, the AllerTop v2.0

[0037] (https: / / www.ddg-pharmfac.net / AllerTOP / ) and ToxinPred2

[0038] (https: / / webs.iiitd.edu.in / raghava / toxinpred2 / index.html) The sensitization and toxicity of epitopes were predicted, and relatively conservative, highly immunogenic, non-toxic, non-sensitizing and hydrophilic T / B cell multi-epitopes were obtained, namely, sequences SEQ ID No. 1-29.

[0039] Conservation analysis: To evaluate the similarity between STEVOR protein and human protein and the potential risk of autoimmune reaction, the four STEVOR sequences were imported into PlasmoDB and the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ) for BLASTP homology sequence alignment; multiple sequence alignment was performed using the ClustalW method, and Weblogo was used to identify the sequences of STEVOR proteins.

[0040] (https: / / weblogo.berkeley.edu / logo.cgi) for amino acid conservation visualization analysis and Shannon Entropy-One

[0041] (https: / / www.hiv.lanl.gov / content / sequence / ENTROPY / entropy.html) to calculate amino acid entropy (see Figure 2 ).

[0042] Fusion epitope construction: To evaluate the immunogenicity of multi-epitope peptides, the selected epitopes were fused to STEVOR proteins into multi-epitope units: ST1-Grp, ST2-Grp, ST3-Grp and ST4-Grp; the fusion epitope construction method was as follows: the multi-epitope peptides selected from each STEVOR group ( Figure 1) were sequentially connected in series to form a multi-epitope construct with the highest antigenicity score to optimize the immune response effect (see Table 1).

[0043] Table 1

[0044]

[0045] In vivo immunogenicity evaluation experiment in BALB / c mice

[0046] The fusion polytope units (i.e., ST1-Grp, ST2-Grp, ST3-Grp, and ST4-Grp) obtained above were verified by computer simulation to be highly immunogenic, non-allergenic, and non-toxic. A B / T cell polytope sequence of Plasmodium falciparum merozoite surface protein 1 (MSP1_p83: FTDPLELEQIPFNLKIRANELDVLKKLV) was used as a positive control. Six-week-old female BALB / c mice were divided into six groups (n=6 per group) and immunized subcutaneously: one adjuvant control group (Freund's adjuvant), four experimental groups (ST1-Grp, ST2-Grp, ST3-Grp, and ST4-Grp fusion polytopes mixed with adjuvant), and one PBS control group (PBS with adjuvant). Immunizations were performed on days 1, 15, 29, and 43, and blood was collected via the tail vein on days 0, 14, 28, 42, and 56. The specific protocol was as follows: the adjuvant control group received an injection of 100 μL of a mixture of PBS and adjuvant emulsion, the PBS control group received an injection of 100 μL of PBS, and each experimental group received an injection of 100 μL of a 0.5 mg / mL mixture of epitopes (ST1-Grp to ST4-Grp) and adjuvant. Except for the PBS and MSP1_p83 control groups, all other groups received Freund's complete adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations. After each blood draw, the sample was allowed to clot at room temperature for 1 hour before serum separation.

[0047] Assessment of immune responses in immunized mice

[0048] The anti-STEVOR specific antibodies in the serum of immunized mice were detected by the aforementioned ELISA method, and HRP-labeled goat anti-mouse IgG was used as the detection antibody (see Figure 3 The levels of circulating cytokines (IFN-γ and IL-2) in serum were quantified using commercial ELISA kits (Mouse IFN gamma Uncoated ELISA Kit and Mouse IL-2 Uncoated ELISA Kit), and the concentrations of each cytokine were calculated by interpolation using the standard curves provided by the kits (see Figure 4 ).

[0049] To assess splenic lymphocyte proliferation, mice were sacrificed by cervical dislocation on day 56. Spleens were aseptically isolated and minced in pre-chilled PBS. After grinding through a 70 μm filter and rinsing with PBS, erythrocytes were lysed with erythrocyte lysis buffer and the rinsing step was repeated to ensure sufficient lysis. Lymphocytes were resuspended and adjusted to a viable cell concentration of 1 × 10^7 / ml. Single fluorescently labeled antibodies (APC-labeled anti-mouse CD4 antibody, Catalog No. 100516; PE / Cyanine 7-labeled anti-mouse CD8a antibody, Catalog No. 100722) were used to stain specific cell surface antigens, and CD4+ and CD8+ T cells were phenotyped using a CytoFLEX S flow cytometer (see ). Figure 5 ).

[0050] Depend on Figure 3 The following are shown: (A) Changes in the average specific IgG antibody levels of mice in each group with increasing number of immunizations. Average specific IgG antibody levels against the fusion multi-epitope peptide increased in all four immunization groups. (B) Results from the fifth blood draw showed that antibody levels in all four immunization groups were higher than those in the PBS and adjuvant control groups (P < 0.05). Among the four immunization groups, the ST1-Grp group had significantly higher antibody levels than the other immunization groups.

[0051] Depend on Figure 4 Visible: IL-2 levels change with increasing number of immunizations (left). (B) IFN-γ levels change with increasing number of immunizations (right). It was found that IL-2 and IFN-γ levels decreased with increasing number of immunizations.

[0052] Depend on Figure 5 Flow cytometry results showed that compared with the PBS and adjuvant control groups, mice immunized with the multi-epitope peptide had significantly higher CD8+ and CD4+ T cell proliferation (P<0.05). The number of CD4+ T cells in mice immunized with the multi-epitope peptide was significantly higher than that in the MSP1_p83-positive control group (P<0.05). Among them, the ST3-Grp group had the highest proportions of CD8+ T cells (19.56%) and CD4+ T cells (54.43%).

[0053] Example 2

[0054] 1) MEFA vaccine construct design:

[0055] To facilitate the screening and optimization of vaccine candidate molecules, a multi-epitope fusion antigen (MEFA) vaccine construct was designed, which integrated the multi-epitope peptide sequences screened from each of the four STEVOR proteins (see Figure 1, Table 1), rather than directly using fused multiple epitopes; the identification of all T cell epitopes was based on the complete human leukocyte antigen (HLA) reference dataset, specifically screening for CD8+ and CD4+ T cell epitopes that overlap or are adjacent to the B cell epitopes in each STEVOR protein (see Table 2); specifically as follows: β-defensin-3 (amino acid sequence: GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK, UniProtKB number: Q5U7J2) was introduced at the first position of the construct as an adjuvant, which can significantly enhance the immunogenicity and efficacy of the multi-epitope vaccine. The specific connection strategy is: use "EAAAK" connecting peptide to connect the adjuvant to CD8+T cell epitopes (SEQID No.1-9), and the epitopes are connected in series through "AAY" connecting peptide; then use "GPGPG" connecting peptide to achieve bridging of CD8+T cell epitopes and CD4+T cell epitopes (SEQ ID No.10-20) and interconnection between CD4+T cell epitopes; finally, use "KK" connecting peptide to connect B cell epitopes (SEQ ID No.21-29) and to CD8+T cell epitopes. It should be noted that each group of epitopes are arranged in ascending order according to the antigenicity score to optimize the immune response. The complete construct structure is: adjuvant (β-defensin-3)-connecting peptide (EAAAK)-CD8+T cell epitope (interconnected by AAY connecting peptide)-connecting peptide (GPGPG)-CD4+T cell epitope (interconnected by GPGPG connecting peptide)-connecting peptide (KK)-B cell epitope (interconnected by KK connecting peptide) (see Figure 6 ).

[0056] Table 2

[0057]

[0058]

[0059] 2)Structure and physical and chemical properties:

[0060] (1) 3D model structure analysis using AlphaFold3 (see Figure 7 ), PROCHECK verification showed that 95% of the amino acid residues were located in the dominant region of the Ramachandran plot, confirming the model's structural stability (-1.169) and good quality (hydrophilicity: 22.61), namely, residues located in the optimal allowed region [A, B, L]; residues located in the additional allowed region [a, b, l, p]; and residues located in the relaxed allowed region [~a, ~b, ~l, ~p].

[0061] (2) The molecular weight of the vaccine construct is 57.02 kDa, and computer simulation predicts that its half-life in mammalian cells is >30 hours.

[0062] 3) Verification experiment

[0063] Computer simulation verification:

[0064] (1) The vaccine constructs were analyzed using ProtParam (https: / / web.expasy.org / protparam / ) for their overall average hydrophilicity (GRAVY), aliphatic index, instability index, and predicted half-life in mammalian cells (i.e., stability: -1.169, hydrophilicity: 22.61), yeast, and Escherichia coli. Key steps included sequence normalization, algorithm selection, and cross-validation of results to ensure that no rare residues (e.g., selenocysteine) interfered with the calculations. 1. Sequence input: Submit the amino acid sequence of the vaccine protein (FASTA format or directly paste); 2. Parameter calculation: (i) GRAVY: Calculate the arithmetic mean of the hydrophilicity scores of all residues based on the Kyte-Doolittle scale (negative value = hydrophilic, positive value = hydrophobic); (ii) Aliphatic index: Assess thermal stability by the relative content of alanine, valine, isoleucine, and leucine (the higher the value, the stronger the stability); (iii) Instability index: Analyze the frequency of unstable motifs (such as polar residue pairs) within a 40-amino acid window (value <40 is considered stable); (iv) Half-life: Predict protein stability in mammals (about 30 hours), yeast (about 20 hours), and Escherichia coli (about 10 hours) based on the N-terminal rule and host metabolic characteristics; 3. Result output: Automatically generate a detailed report containing all parameters. AllerTop v2.0 (https: / / www.ddg-pharmfac.net / AllerTOP / ) and ToxinPred2 (https: / / webs.iiitd.edu.in / raghava / toxinpred2 / ) were used to assess allergenicity and toxicity (i.e., to obtain non-toxic and non-allergenic properties). The steps are as follows: Sequence submission: Input the amino acid sequence of the vaccine protein (FASTA format or directly paste); AllerTop analysis: Based on the k-NN algorithm, sequence features (such as dipeptide frequency and cysteine ​​content) are evaluated and the output is "allergen" or "non-allergen" classification (with an accuracy rate >85%); ToxinPred 2 uses a support vector machine (SVM) model to analyze toxicity-related motifs (such as hemolytic peptide characteristics), physicochemical properties (such as hydrophobicity), and sequence similarity (compared to a database of known toxins) to output a toxicity score (a threshold >0.5 is considered potentially toxic).

[0065] (2) The multi-epitope fusion antigen (MEFA) vaccine construct was recombinantly expressed using an E. coli expression system: First, the STVC gene (537 amino acids, with a 6×His tag at the N-terminus) containing a His tag was transferred into E. coli, and the target protein was obtained after induction of expression. The expression product was obtained by lysing the bacterial supernatant and then purified by nickel column affinity chromatography. Finally, a total weight of 11.48 mg of protein was obtained with a concentration of 0.82 mg / ml (Bradford method) and a purity of ≥90% (SDS-PAGE reducing conditions detection) (see Figure 8 ).

[0066] Depend on Figure 8 Mass spectroscopy analysis revealed clear bands under reducing conditions on SDS-PAGE (confirmed molecular weight by comparison with a marker). Western blot analysis confirmed specific binding of the His-tagged protein using a mouse anti-His monoclonal antibody (GenScript, model A00186). The purified protein was stored in PBS (pH 7.4) at -80°C to avoid repeated freeze-thaw cycles. Key experimental parameters (e.g., marker [Bio-Rad, model 1610374S / GenScript, model M00673], BSA control 2.00 μg) were recorded in the protocol.

[0067] (3) Immunological simulations using C-ImmSim (https: / / kraken.iac.rm.cnr.it / C-IMMSIM / index.php) showed that the vaccine construct could effectively stimulate significant immune responses of T cells and B cells (see Figure 9 The steps of immune simulation of the vaccine construct candidate molecule are as follows:

[0068] 1. Parameter setting: input the vaccine sequence and select the host HLA type (such as HLA-A*02:01); set the injection plan (dose, interval time and number of times, such as 1000 particles, day 0 / 28 / 56); 2. Simulation operation: simulate the innate / adaptive immune response based on the intelligent agent model, including antigen presentation, T / B cell activation and cytokine dynamics; 3. Result analysis: extract key indicators (antibody titer, memory cell number, IFN-γ peak, etc.).

[0069] Depend on Figure 9As can be seen, all three simulated doses of this vaccine construct stimulated significant T and B cell immune responses. The third simulated dose produced a stronger immune response than the first two injections. Total B cell counts peaked around day 60, with memory B cell counts reaching 580 / mm³ at day 60 and remaining at a high level of 410 / mm³ after 350 days. CD8+ T cells peaked at 1080 / mm³ 13 days after injection. Combined immunoglobulin M (IgM) and IgG levels peaked around day 65, at approximately 225,700, then gradually declined to approximately 23,000 by day 350. Memory CD4+ T cell counts peaked at 1890 / mm³ and non-memory CD4+ T cell counts peaked after the third and second injections, at 11,330 / mm³. The IFN-γ response was significantly enhanced after each simulated injection, reaching a peak of 427,000 ng / mL. IL-2 concentrations reached 478,000 ng / mL 60 days after injection.

[0070] Sequence Listing:

[0071] Adjuvant (β-defensin-3), "EAAAK" connecting peptide, CD8+T cell epitope: SEQ ID No.1-9 ("AAY" connecting peptide is the interconnection between epitopes), "GPGPG" connecting peptide, CD4+T cell epitope: SEQ ID No.10-20 ("GPGPG" connecting peptide is the interconnection between epitopes), "KK" connecting peptide, B cell epitope: SEQ ID No.21-29 ("KK" connecting peptide is the interconnection between epitopes).

[0072] β-Defensin-3:

[0073] GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK CD8+ T cell epitope:

[0074] SEQ ID No.1:HAMLKSGRY

[0075] SEQ ID No.2: EIFGNESDM

[0076] SEQ ID No.3:KSRLLAQTQ

[0077] SEQ ID No.4: VFGDKNHAM

[0078] SEQ ID No.5:DVFGDKNHA

[0079] SEQ ID No.6:RLLAQTQNH

[0080] SEQ ID No.7: NTHHPYKQL

[0081] SEQ ID No.8: NDAEPISTL

[0082] SEQ ID No.9: VEKNVTKHV

[0083] CD4+ T cell epitope:

[0084] SEQ ID No.10: QGTTIKSRLLAQTQN

[0085] SEQ ID No.11: GTTIKSRLLAQTQNH

[0086] SEQ ID No.12: IKSRLLAQTQNHNPH

[0087] SEQ ID No.13: TIKSRLLAQTQNHNP

[0088] SEQ ID No.14: KSRLLAQTQNHNPHY

[0089] SEQ ID No.15: YEDVFGDKNHAMLKS

[0090] SEQ ID No.16: EKYEDVFGDKNHAML

[0091] SEQ ID No.17: VVEKNGTKIRGGNSA

[0092] SEQ ID No.18: QNHNPHYHNDPELKE

[0093] SEQ ID No.19: THDPYKQLKEVVEKN

[0094] SEQ ID No.20: THHPYKQLKEVVEKN

[0095] B cell epitope:

[0096] SEQ ID No.21: EEIFGNESDMLKSGMSPNVD

[0097] SEQ ID No.22: EDVFGDKNHAMLKSGRYPNDDDESDDS

[0098] SEQ ID No.23: EKNGTKYSGGNDAEP

[0099] SEQ ID No.24:IKKYQNT

[0100] SEQ ID No.25:TKHVGGNDTEP

[0101] SEQ ID No.26:TKPVGEHGTEP

[0102] SEQ ID No.27:QLKEVVEKNGTKIRGGNSAE

[0103] SEQ ID No.28: IKKYQQT

[0104] SEQ ID No.29:QNHNPHYHND

[0105] Total length (adjuvant, epitope, linker): 522 amino acids

[0106] Type: Amino acid sequence

[0107] Artificial Sequence

[0108] GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKKEAAA

[0109] KHAMLKSGRYAAYEIFGNESDMAAYKSRLLAQTQAAYVFGDKNHAMA

[0110] AYDVFGDKNHAAAYRLLAQTQNHAAYNTHHPYKQLAAYNDAEPISTLA

[0111] AYVEKNVTKHVGPGPGQGTTIKSRLLAQTQNGPGPGGTTIKSRLLAQTQ

[0112] NHGPGPGIKSRLLAQTQNHNPHGPGPGTIKSRLLAQTQNHNPGPGPGKS

[0113] RLLAQTQNHNPHYGPGPGYEDVFGDKNHAMLKSGPGPGEKYEDVFGD

[0114] KNHAMLGPGPGVVEKNGTKIRGGNSAGPGPGQNHNPHYHNDPELKEGP

[0115] GPGTHDPYKQLKEVVEKNGPGPGTHHPYKQLKEVVEKNKKEEIFGNES

[0116] DMLKSGMSPNVDKKEDVFGDKNHAMLKSGRYPNDDDESDDSKKEKN

[0117] GTKYSGGNDAEPKKIKKYQNTKKTKHVGGNDTEPKKTKPVGEHGTEPK

[0118] KKQLKEVVEKNGTKIRGGNSAEKKIKKYQQTKKQNHNPHYHND

[0119] In summary, the present invention obtains immune antigens and constructs a multi-epitope fusion antigen vaccine. Analysis confirms that the construct is stable (95% of amino acid residues are located in the Ramachandran map dominant region), non-allergenic, and has a global HLA coverage rate of 97.15%. In vivo experiments show that immunized mice can produce high-titer IgG antibodies and promote T cell proliferation. Clinical sera from malaria-endemic areas show high reactivity to the fusion epitope. Therefore, the vaccine can be produced on a large scale and is cost-effective, making it particularly suitable for use in high-transmission areas.

Claims

1. A multi-epitope fusion antigen vaccine derived from the STEVOR protein of Plasmodium falciparum, characterized by: The multi-epitope fusion antigen vaccine contains at least two amino acid fragments of B cell epitopes, CD4 and T cell epitopes, and CD8 and T cell epitopes.

2. The multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum according to claim 1, characterized in that: The multi-epitope fusion antigen vaccine contains at least two of the amino acid sequences described in SEQ ID No: 1-29.

3. The multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum according to claim 2, characterized in that: The multi-epitope fusion antigen vaccine is the amino acid sequence of SEQ ID No: 1-29 and is connected by a connecting peptide.

4. The multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum according to claim 3, characterized in that: The multi-epitope fusion antigen vaccine is an amino acid sequence as described in SEQ ID No: 1-29 and is connected to a β-defensin-3 adjuvant via a connecting peptide; wherein the β-defensin-3 adjuvant amino acid sequence is shown in SEQ ID No:

30.

5. The multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum according to claim 4, characterized in that: The amino acid sequences of the CD8 and T cell epitopes are SEQ ID Nos: 1-9; the amino acid sequences of the CD4 and T cell epitopes are SEQ ID Nos: 10-20; and the amino acid sequences of the B cell epitopes are SEQ ID Nos: 21-29.

6. The multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum according to any one of claims 1 to 5, characterized in that: The multi-epitope fusion antigen vaccine is composed of an adjuvant (β-defensin-3), a connecting peptide (EAAAK), CD8 and T cell epitope amino acid sequences, a connecting peptide (GPGPG), CD4 and T cell epitope amino acid sequences, a connecting peptide (KK), and a B cell epitope amino acid sequence.

7. A method for constructing the multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum according to claim 1, characterized in that: The adjuvant was connected to the CD8+T cell epitope (SEQ ID No. 1-9) using a rigid connecting peptide, and then the CD8+T cell epitope and the CD4+T cell epitope (SEQ ID No. 10-20) were bridged by the "GPGPG" connecting peptide; the B cell epitope (SEQ ID No. 21-29) was connected to the CD4+T cell epitope through a connecting peptide (KK), and then the sequences in the B cell epitope were interconnected through the KK connecting peptide.

8. The method for constructing the multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum according to claim 7, characterized in that: The sequences in the CD8+T cell epitopes (SEQ ID No. 1-9) are interconnected through an AAY connecting peptide; the sequences in the CD4+T cell epitopes (SEQ ID No. 10-20) are interconnected through a GPGPG connecting peptide; and the sequences in the B cell epitopes (SEQ ID No. 21-29) are interconnected through a KK connecting peptide.

9. A use of the multi-epitope fusion antigen vaccine derived from STEVOR protein of Plasmodium falciparum according to claim 1, characterized in that: The vaccine is used as a malaria vaccine.

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