A polyepitope fusion protein, gene, vaccine for preventing bungomavirus and preparation method thereof

Abstract

The application discloses a kind of for preventing Bungowenia virus Polyepitope fusion protein, gene, vaccine and preparation method thereof, belong to biological medicine technical field.The Polyepitope fusion protein for preventing Bungowenia virus provided in the application is obtained in series by E. coli outer membrane anchoring protein ClyA, four MHC I class restricted T cell epitopes, four MHC II class restricted T cell epitopes, two B cell epitopes and BALB / c mouse immunoglobulin Fc fragment.The vaccine of the application is prepared by high-pressure homogenization process to obtain the engineered artificial nanovesicle (E-aBNV) with high-density surface display antigen array.The E-aBNV utilizes itself pathogen-associated molecular pattern to provide "self-adjuvant" effect, can efficiently activate B cell receptor and promote antigen cross-presentation, thereby synergistically inducing high level of neutralizing antibody and Th1-biased cellular immunity, which can effectively block the infection of Bungowenia virus to host cell.

Description

Technical Field

[0001] This invention relates to a multi-epitope fusion protein, gene, vaccine, and preparation method for preventing chikungunya virus, belonging to the field of biomedical technology. Background Technology

[0002] Chikungunya virus is an arbovirus transmitted by Aedes mosquitoes, belonging to the genus Alphavirus of the family Pseudaviviridae. It was first isolated in Tanzania in 1952. The virus has now spread to more than 100 countries and regions worldwide, with endemic areas covering tropical and subtropical regions of Africa, Asia, the Americas, and parts of Europe. Approximately 16.9 million new infections are reported annually, putting nearly 2.8 billion people at risk of infection. Clinical features include sudden onset of high fever, rash, and severe polyarthritis, with the acute phase typically lasting 2 to 3 weeks. Some patients may develop chronic arthritis, with symptoms persisting for months or even years, leading to decreased mobility and impaired quality of life. In recent years, with global warming and accelerated urbanization, the distribution range of mosquito vectors has continued to expand, significantly increasing the risk of chikungunya virus transmission. The direct medical costs and indirect social costs of this disease are both high, posing a significant public health burden in resource-limited areas.

[0003] Currently, there are three main types of approved chikungunya virus vaccines. Live attenuated vaccines offer single-dose immunization and strong immunogenicity, but they carry a risk of virulence reversion in the elderly and immunocompromised individuals, potentially inducing vaccine-associated arthritis, fever, and other adverse reactions. Some products have had their clinical application suspended due to safety concerns. Virus-like particle vaccines do not contain viral genetic material and have relatively good safety profiles, but multiple doses are required to maintain immune protection, and antibody titers tend to decline over time, requiring improvement in the durability of protection. Furthermore, the production process for this type of vaccine involves eukaryotic expression systems, resulting in high costs and pricing far exceeding the affordability of low- and middle-income countries. While messenger RNA (mRNA) vaccines have shown good immunogenicity in early clinical trials, they are highly dependent on ultra-low-temperature cold chain storage and transportation, making stable supply difficult to achieve in tropical regions and primary healthcare facilities. In summary, existing vaccine platforms have varying degrees of limitations in terms of safety, efficacy, stability, and production costs, making it difficult to fully meet the prevention and control needs of epidemic areas worldwide. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a multi-epitope fusion protein for the prevention of chikungunya virus, as well as its gene, vaccine and preparation method.

[0005] Technical Solution: The present invention describes a multi-epitope fusion protein for preventing Chikungunya virus. The fusion protein comprises the E. coli outer membrane anchoring protein ClyA as shown in SEQ ID NO.1, four MHC class I restricted T cell epitopes as shown in SEQ ID NO.2-5, four MHC class II restricted T cell epitopes as shown in SEQ ID NO.6-9, two B cell epitopes as shown in SEQ ID NO.10-11, and the BALB / c mouse immunoglobulin Fc fragment as shown in SEQ ID NO.12, which are tandemly linked by flexible linkers.

[0006] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO.13.

[0007] The gene encoding the aforementioned multi-epitope fusion protein for preventing Chikungunya virus is described in this invention.

[0008] Furthermore, the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO.14.

[0009] The recombinant expression vector of the present invention contains the gene for the multi-epitope fusion protein used to prevent chikungunya virus.

[0010] The genetically engineered bacteria of this invention are produced by transforming the above-mentioned recombinant expression vector into... ClearColi BL21 (DE3) Obtained from strains.

[0011] The present invention relates to the application of the multi-epitope fusion protein for preventing Chikungunya virus, the gene, the recombinant expression vector, and the genetically engineered bacteria in the preparation of drugs for preventing Chikungunya virus infection.

[0012] The chikungunya virus vaccine of the present invention comprises engineered nanovesicles obtained by high-pressure homogenization of the above-mentioned genetically engineered bacteria.

[0013] The method for preparing the chikungunya virus vaccine of the present invention includes the following steps: ligating a gene fragment with a nucleotide sequence as shown in SEQ ID NO. 14 to a basic vector and then transforming it into Escherichia coli. ClearColi BL21(DE3) Competent cells were induced to express IPTG; the bacteria were homogenized by high pressure and purified by differential centrifugation.

[0014] Furthermore, the high-pressure homogenization conditions were 800 bar and 10 cycles.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention constructs a chikungunya virus vaccine based on bacterial nanovesicle delivery of multi-epitope antigens through immunoinformatics, artificial intelligence-assisted structure prediction and genetic engineering technology. In BALB / c mice, it can induce the production of neutralizing antibodies and Th1-biased cellular immune responses, and can effectively block the infection of host cells by chikungunya virus. Attached Figure Description

[0016] Figure 1 This is the structure of the fusion protein in Example 1 of the present invention and its interaction with the receptor, where a is a schematic diagram of the structure of the ClyA-multi-epitope-mFc fusion protein, and b is a molecular docking analysis diagram of the antigen epitope region and the receptor MXRA8; Figure 2 This is the engineering implementation in Embodiment 1 of the present invention. ClearColi BL21(DE3) Immunoblot diagram of the strain expressing the above fusion protein; Figure 3 These are electron microscope images of E-OMV and E-aBNV in Embodiment 1 of the present invention; Figure 4 This is a statistical chart of the particle size and zeta potential of E-OMV and E-aBNV in Example 1 of the present invention; Figure 5 This is a protein immunoblot map of the fusion protein expressed by E-OMV and E-aBNV in Example 1 of the present invention; Figure 6 This is a particle size distribution chart of E-aBNV stored at different temperatures for 7 days in Example 1 of this invention; Figure 7 This is a production statistics chart of OMV, aBNV, E-OMV- and E-aBNV in Embodiment 1 of the present invention; Figure 8 These are electron microscope images and particle size statistics of E-aBNV after lyophilization and reconstitution in Example 1 of this invention; Figure 9 These are representative flow cytometry plots and statistical graphs of cell apoptosis after co-incubation of E-aBNV and DC2.4 cells obtained in the flow cytometry experiment of Example 2 of the present invention; Figure 10 These are the immunofluorescence colocalization map, flow cytometry representation map, and statistical graph of DC2.4 uptake of E-aBNV in Example 2 of the present invention; Figure 11 These are flow cytometry representations and statistical plots of E-aBNV-stimulated activation of bone marrow-derived dendritic cells (BMDCs) in Example 2 of this invention; Figure 12This is a statistical chart showing the levels of TNF-α, IL-1β, IL-6, and IL-12p70 after co-incubation of E-aBNV with BMDCs in Example 2 of this invention. Figure 13 This is a flow cytometry plot of the specific immune response induced by the stimulated bone marrow-derived dendritic cells in naïve T cells in Embodiment 2 of the present invention. Figure 14 This is a fluorescence imaging image after intramuscular injection of E-aBNV in Example 3 of the present invention, where a is an in vivo fluorescence imaging image of biological distribution in vivo, and b is an in vitro fluorescence imaging image and statistical diagram of each organ. Figure 15 This is a representative diagram and weight statistics of mouse inguinal lymph nodes in Example 3 of the present invention; Figure 16 In Example 3 of this invention, the activation analysis of dendritic cells (DCs) stimulated by E-aBNV is shown, where a is an immunofluorescence colocalization map of DCs uptake of E-aBNV in lymph nodes, and b is a flow cytometry representation and statistical graph of DC activation in lymph nodes. Figure 17 This is an immunofluorescence image of E-aBNV stimulating the formation of germinal centers in lymph nodes in Example 4 of this invention; Figure 18 These are flow cytometry representations and statistical graphs of inguinal lymph node follicular helper T cells (Tfh) obtained in the flow cytometry experiment in Example 4 of this invention. Figure 19 These are representative flow cytometry plots and statistical graphs of inguinal lymph node B cells and germinal centers obtained in the flow cytometry experiment of Example 4 of this invention. Figure 20 This is a statistical chart showing the titers of specific IgG antibodies against E1 and E2 antigenic epitopes in the serum of immunized mice in Example 4 of this invention, as well as the titers of pseudovirus neutralizing antibodies. Figure 21 It is the CD4 of the spleen of the immunized mouse in Example 5 of this invention. + and CD8 + Flow cytometry statistics of T cell activation and memory cell typing; Figure 22 The specific CD4 in the spleen of immunized mice in Example 5 of this invention + and CD8 + Flow cytometry plot of T cells; Figure 23 This is a statistical chart of blood biochemistry and routine blood indicators of serum from mice that were immunized for short and long periods of time in Example 6 of this invention; Figure 24 This is a statistical chart of the weight changes of mice during the immunization process in Example 6 of the present invention; Figure 25These are H&E slices of major organs and tissues from mice subjected to short-term and long-term immunization in Example 6 of this invention. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0018] Example 1: Preparation of multi-epitope engineered bacterial nanovesicles (E-aBNV) (1) Immunoinformatics design and screening of multi-epitope antigens Based on the conserved sequences of E1 and E2 proteins of 769 identified chikungunya virus strains, the IEDB analysis resource database was used to predict H-2d restricted T cell epitopes and B cell linear epitopes applicable to BALB / c mice. From these, four MHC class I restricted T cell epitopes with the highest affinity sequences (SEQ ID NO. 2-5), four MHC class II restricted T cell epitopes (SEQ ID NO. 6-9), and two B cell linear epitopes (SEQ ID NO. 10-11) were screened. Each epitope is tandemly linked by a 5×Gly-Ser flexible linker and then linked to the E. coli outer membrane anchoring protein ClyA (amino acid sequence as shown in SEQ ID NO.1) and the BALB / c mouse mFc fragment (amino acid sequence as shown in SEQ ID NO.12). The order from N-terminus to C-terminus is as follows: ClyA (SEQ ID NO.1) — B cell epitope (SEQ ID NO.10) — MHC I epitope (SEQ ID NO.2) — MHC II epitope (SEQ ID NO.6) — MHC I epitope (SEQ ID NO.3) — MHC II epitope (SEQ ID NO.7) — B cell epitope (SEQ ID NO.11) — MHC I epitope (SEQ ID NO.4) — MHC II epitope (SEQ ID NO.8) — MHC I epitope (SEQ ID NO.5) — MHC II epitope (SEQ ID NO.9) — mFc (SEQ ID NO.12). The amino acid sequence of the recombinant protein is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.14. Figure 1 The three-dimensional structure of the fusion protein was predicted using AlphaFold3, and molecular docking analysis verified that the antigen epitope region and the receptor MXRA8 have a good binding ability.

[0019] SEQ ID NO.1:MTEIVADKTVEVVKNAIETADGALDLYNKYLDQVIPWQTFDETIKELSRFKQEYSQAASVLVGDIKTLLMDSQDKYFEATQTVYEWCGVATQLLAAYILLFDEYNEKKASAQKDILIKVLDDGITKLNEAQKSLLVSSQSFNNASGKLLALDSQLTNDFSEKSSYFQSQVDKIRKEAYAGAAAGVVAGPFGLIISYSIAAGVVEGKLIPELKNKLKSVQNFFTTLSNTVKQANKDIDAAKLKLTTEIAAIGEIKTETETTRFYVDYDDLMLSLLKEAAKKMINTCNEYQKRHGKKTLFEVPEV SEQ ID NO.2:TYQEAAVYL SEQ ID NO.3:SPMVLEMEL SEQ ID NO.4:YYYELYPTM SEQ ID NO.5:TPGATVPFL SEQ ID NO.6:SQLQISFSTALASAE SEQ ID NO.7:SAYRAHTASASAKLR SEQ ID NO.8:KWQYNSPLVPRNAEL SEQ ID NO.9:YYELYPTMTVVVVSV SEQ ID NO.10:VYNMDYPPFGAGRPGQFGDIQSRTPESEDVY SEQ ID NO.11:YVQSTAATTEEIEVHMPPDTPDRTLMSQQSGN SEQ ID NO.12:VPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO.13: MTEIVADKTVEVVKNAIETADGALDLYNKYLDQVIPWQTFDETIKELSRFKQEYSQAASVLVGDIKTLLMDSQDKYFEATQTVYEWCGVATQLLAAYILLFDEYNEKKASAQKDILIKVLDDGITKLNEAQKSLLVSSQSFNNASGKLLALDSQLTNDFSEKSSYFQSQVDKIRKEAYAGAAAGVVAGPFGLIISYSIAAGVVEGKLIPELKNKLKSVQNFFTTLSNTVKQANKDIDAAKLKLTTEIAAIGEIKTETTRFYVDYDDLMLSLLKEAAKKMINTCNEYQKRHGKKTLFEVPEVGGGGSVYNMDYPPFGAGRPGQFGDIQSRTPESEDVYGGGGSTYQEAAVYLGGGGSSQLQISFSTAL ASAEGGGGSSPMVLEMELGGGGSSAYRAHTASASAKLRGGGGSYVQSTAATTEEIEVHMPPDTPDTRTLMSQQSGNGGGGSYYYELYPTMGGGGSKWQYNSPLVPRNAELGGGGSTPGATVPFLGGGGSYYELYPTMTVVVVSVGGGSVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSGPK (2) Construction of engineered Escherichia coli The sequence of the fusion protein was obtained through gene synthesis, and after double digestion with BamHI and XhoI, it was cloned into the pET-28a vector (purchased from Sangon Biotech, B540183) to obtain the recombinant plasmid pET28a-ClyA-epitopes-mFc. Subsequently, the recombinant plasmid pET28a-ClyA-epitopes-mFc was heat-shock transformed into Escherichia coli. ClearColi BL21(DE3) Competent cells (purchased from Lucigen, 608101) were plated on LB agar plates containing kanamycin (50 μg / mL) and cultured at 37°C for 18-24 hours. Single colonies were picked and inoculated into LB liquid medium containing kanamycin and cultured at 37°C and 220 rpm until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.5 mM, and expression was induced for 16 hours at 20°C and 120 rpm to obtain engineered *E. coli*. Bacterial cells were collected for Western blotting verification, such as... Figure 2 As shown, a specific band of approximately 80 kDa was detected using anti-6×His antibody, consistent with the expected molecular weight.

[0020] (3) Preparation of engineered artificial outer membrane vesicles (E-aBNV) Engineered *E. coli* were inoculated into LB medium and cultured at 37°C and 220 rpm until an OD600 of 0.6–0.8 was reached. IPTG was added to a final concentration of 0.5 mM, and expression was induced for 16 hours at 20°C and 120 rpm. Cells were collected by centrifugation at 8000 rpm for 10 minutes, washed twice with PBS, and resuspended in PBS. Mechanical disruption was performed using a high-pressure homogenizer at 4°C, 800 bar, and 10 cycles. The disruption buffer was centrifuged at 8000 rpm for 30 minutes to remove cell debris. The supernatant was filtered through a 0.45 μm filter and then concentrated using a 100 kDa ultrafiltration tube. The concentrate was ultracentrifuged at 150,000 × g for 1 hour, the precipitate was resuspended in sterile PBS, and finally sterilely filtered through a 0.22 μm filter to obtain the E-aBNV suspension. E-OMV (naturally secreted vesicles) from engineered E. coli were prepared as a control. E-OMV was spontaneously produced by engineered E. coli during culture and was obtained by differential centrifugation and ultracentrifugation without high-pressure homogenization.

[0021] (4) Characterization of E-aBNV Vesicle morphology was observed using a transmission electron microscope (TEM, model: FEI TALOS 200×), such as... Figure 3As shown, both E-OMV and E-aBNV exhibit a uniform spherical structure. Particle size distribution and zeta potential were determined using a dynamic light scattering spectrometer (DLS, model: Nano ZS90), and the results are as follows. Figure 4 As shown, there was no significant difference between E-aBNV and E-OMV; both had an average particle size of approximately 140 nm and a zeta potential of approximately -10 ± 3 mV. Western blotting was used to verify the expression of the fusion protein in the vesicles using an anti-6×His antibody, and the results are as follows: Figure 5 As shown, both the E-aBNV and E-OMV groups exhibited specific bands at 80 kDa.

[0022] (5) Stability assessment of E-aBNV To evaluate the stability of E-aBNV under different storage conditions, E-aBNV was stored at 4℃, -20℃, and -80℃ for 7 days, and its particle size change was measured daily. The results are as follows: Figure 6 As shown, the particle size change in each temperature group was <10%, indicating that E-aBNV has good temperature stability. Further, E-aBNV was freeze-dried to prepare a stable powder, and OMV (initial Escherichia coli) was analyzed. ClearColi BL21(DE3) Naturally secreted outer membrane vesicles), E-OMV (engineered E. coli) ClearColi BL21(DE3) Naturally secreted outer membrane vesicles), E-aBNV (engineered E. coli) ClearColi BL21 (DE3) Nanovesicles prepared by high-pressure homogenization), aBNV (initial Escherichia coli) ClearColi BL21(DE3) Yield analysis was performed on nanovesicles prepared by high-pressure homogenization. The yield analysis showed that the yield of artificially prepared BNVs was approximately 100 times that of naturally secreted OMVs (e.g., ...). Figure 7 (As shown). To evaluate the effect of lyophilization on the morphology of E-aBNV, the lyophilized formulation was reconstituted and observed by transmission electron microscopy and dynamic light scattering measurement. The results are shown below. Figure 8 As shown, the reconstituted E-aBNV (FD-E-aBNV) maintains a uniform spherical structure with an average particle size of about 140 nm, which is not significantly different from that before lyophilization, confirming that the formulation has excellent lyophilization stability and reconstitution properties.

[0023] Example 2: In vitro immune activation of E-aBNV (1) Analysis of antigen delivery kinetics E-aBNV cells were co-incubated with DC2.4 cells for 48 hours, and the cells were stained with Annexin V / PI. Flow cytometry analysis results are as follows: Figure 9As shown, E-aBNV did not induce apoptosis or necrosis within the tested concentration range (10-30 μg / mL), confirming its good cell safety. Further, FITC-labeled E-aBNV was co-incubated with DC2.4 cells for 12 hours. Cell nuclei were labeled with Hoechst (blue), and lysosomes were labeled with LysoTracker-Red (red). Confocal microscopy observations are shown below. Figure 10 As shown, after 12 hours, E-aBNV was digested in lysosomes, and the antigen was released from the lysosomes. Flow cytometry analysis also confirmed the efficient uptake of E-aBNV by DC2.4 cells, laying the foundation for subsequent antigen processing, presentation, and immune activation.

[0024] (2) Effects on dendritic cell maturation Bone marrow-derived dendritic cells (BMDCs) were co-incubated for 24 hours with PBS, 20 μg / mL aBNV + 10 free antigenic peptides (i.e., epitope peptides shown in SEQ ID NO. 2~11, 1 μg / mL each), 10 free antigenic peptides (1 μg / mL each), and 20 μg / mL LE-aBNV. Cells were collected and analyzed by flow cytometry. The results are as follows: Figure 11 As shown, compared with the PBS group and the peptide group, both the aBNV+ peptide group and the E-aBNV group significantly upregulated the expression levels of co-stimulatory molecules CD80, CD86, MHC1, and MHC II on the surface of BMDCs. Culture supernatant was collected, and cytokine secretion was detected using ELISA. The results are shown below. Figure 12 As shown, the levels of TNF-α, IL-1β, IL-6, and IL-12p70 in the E-aBNV group and the aBNV+ peptide group were significantly higher than those in the PBS group and the peptide group, indicating that E-aBNV and aBNV+ peptide can effectively activate dendritic cells and promote the secretion of pro-inflammatory cytokines.

[0025] (3) Inducing specific T cell immune responses The stimulated BMDCs were co-cultured with naive T cells for 24 hours, and the cells were collected for flow cytometry analysis. The results are as follows: Figure 13 As shown, BMDCs stimulated only by E-aBNV and aBNV+ peptides were able to induce specific T cell immune responses and secrete CD4+ IFN-γ and IL-2. + and CD8 + The proportion of T cells increased significantly, while CD4 cells secreting IL-4 increased. + The proportion of T cells did not change significantly. These results confirm that E-aBNV induces Th1-biased cellular immune responses by activating BMDCs and effectively promotes the generation of antigen-specific cytotoxic T cell responses.

[0026] Example 3: Distribution and Immune Activation of E-aBNV in Vivo (1) Distribution in mice E-aBNV was labeled with DiD fluorescent dye and immunized with BALB / c mice (8-week-old females, approximately 20 g in weight, purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) via intramuscular injection in the leg, with a dose of 100 μg per mouse. Free DiD was used as a control. Observations were performed using a small animal in vivo imaging system at 0, 0.5, 2, 8, 12, and 24 hours. Results are as follows: Figure 14 As shown in figure a, E-aBNV gradually diffuses throughout the body after intramuscular injection in the leg, exhibiting a more persistent retention in the muscle tissue on the injection side. Mice were sacrificed after 12 hours, and major organs were collected for in vitro imaging. The results are as follows: Figure 14 As shown in b, E-aBNV was mainly distributed in the inguinal lymph nodes (iLNs) on the injection side and the muscles at the injection site, with no obvious signal in other organs. The lymph node volume and weight were measured, and the results are as follows: Figure 15 As shown, the volume and weight of iLNs on the injection side were significantly larger than those on the contralateral side (injected with the same amount of PBS), indicating that E-aBNV can effectively migrate to the draining lymph nodes and activate them.

[0027] (2) Activation of lymph node immune cells iLNs from the injection side were collected, prepared into frozen sections, and labeled with DAPI (blue), E-aBNV (red), and CD11c using DiD. + Labeled DCs (green), immunofluorescence staining results as follows Figure 16 As shown, E-aBNV and DCs exhibited significant co-localization. Single-cell suspensions of the injected iLNs were prepared for flow cytometry analysis. Results are as follows... Figure 16 As shown in a, E-aBNV can be taken up by lymph node DCs. Further analysis of DC maturity markers yielded the following results. Figure 16 As shown in b, the expression levels of CD80, CD86, MHC I, and MHC II were significantly upregulated in the E-aBNV group. These results indicate that E-aBNV can migrate to draining lymph nodes after intramuscular injection, and be taken up by DCs and induced to mature, providing the necessary conditions for subsequent antigen presentation and the initiation of adaptive immune responses.

[0028] Example 4: E-aBNV-induced germinal center response and antibody production (1) Formation of the germinal centers of lymph nodes Eight-week-old female BALB / c mice were randomly divided into four groups of six each: PBS group (100 μL), free polypeptide group (containing 10 antigenic polypeptides, 5 μg each, based on protein content), aBNV group (100 μg, based on protein content), and E-aBNV group (100 μg, based on protein content). Three immunizations were administered intramuscularly on days 0, 14, and 28. Ten days after the first immunization, inguinal lymph nodes (iLNs) from the injection side were collected for frozen sectioning, and germinal center formation was detected using immunofluorescence. Immunofluorescence staining was performed using IgD (green), Ki67 (blue), CD35 (white), and CD3 (magenta) antibodies. Results are as follows. Figure 17 As shown, the E-aBNV group germinal centers (Ki67) + CD35 + The number and area of ​​E-aBNV cells were significantly greater than those in the PBS group. This indicates that E-aBNV can effectively activate B cell responses and promote germinal center formation.

[0029] (2) Tfh cell and B cell responses Mice were sacrificed 14 days after the third immunization, and iLNs were collected to prepare single-cell suspensions. Flow cytometry was used to analyze follicular helper T cells (Tfh) and B cell subsets. The Tfh analysis results are as follows: Figure 18 As shown, Tfh cells (CD3) in the E-aBNV group and the aBNV group + CD4⁺CXCR5 + The proportion of Tfh cells was significantly increased compared to other groups, and the expression levels of activation marker ICOS and inhibitory receptor PD-1 were significantly upregulated, indicating that the aBNV vector can effectively promote Tfh cell differentiation and activation. B cell analysis results are as follows... Figure 19 As shown, the total B220⁺ B cells and germinal center B cells (B220) in the E-aBNV group and the aBNV group + GL7 + CD95 + The proportions of the aBNV-vectored nanovaccines were significantly higher than those of other groups, indicating that the nanovaccines could enhance Tfh-B cell interactions and drive the formation of germinal centers.

[0030] (3) The production of specific antibodies and neutralizing antibodies Peripheral blood was collected from mice on day 14 after each immunization. After standing at room temperature for 2 hours, serum was separated by centrifugation at 3000 rpm for 10 minutes. The titers of anti-E1 and anti-E2 specific IgG antibodies in the serum were detected using ELISA. Results are as follows: Figure 20As shown, antibody levels were low in all experimental groups after the initial immunization; after the second immunization, the antibody titer in the E-aBNV group increased significantly; after the third immunization, the antibody titer in this group reached its peak and was significantly higher than that in the other groups. To further evaluate the functional activity of the induced antibodies, a neutralization assay was performed using chikungunya virus pseudovirus. 1.0 × 10 5 TU pseudovirus (purchased from Fosun Pharma, FNV8466) was co-incubated with serum for 1 hour, then transferred to HEK293T cells for further incubation for 72 hours to determine infection efficiency. Results are as follows... Figure 20 As shown, the serum from the E-aBNV group exhibited potent neutralizing activity. These results indicate that the antibodies induced by the E-aBNV vaccine not only possess high specificity but also significant neutralizing capacity, effectively blocking chikungunya virus infection of host cells.

[0031] Example 5: E-aBNV-induced T-cell immune response (1) T cell activation and differentiation Spleens were collected 14 days after the third immunization and prepared into a single-cell suspension. Flow cytometry was used to analyze T cell activation and memory subsets, and the results are as follows: Figure 21 As shown, E-aBNV group CD4 + and CD8 + The expression of CD69, a marker of T cell activation, was significantly upregulated. This confirms that the vaccine can effectively activate T cell immune responses. Further analysis of memory T cell subsets revealed that naive T cells (CD62L) in the E-aBNV group... + CD44 - The proportion of effector memory T cells (Tem, CD62L) decreased significantly, while the proportion of effector memory T cells (Tem, CD62L) decreased significantly. - CD44 + ) and central memory T cells (Tcm, CD62L) + CD44 + The proportion of E-aBNV increased significantly, and the magnitude of the change was greater than that of other groups, indicating that E-aBNV can effectively promote T cell activation and memory differentiation.

[0032] (2) Antigen-specific T cell response Spleen cells were stimulated in vitro for 24 hours with 10 E1 / E2 antigenic epitope peptides (5 μg / mL for each peptide), and intracellular cytokine secretion was detected by flow cytometry. Results are as follows: Figure 22 As shown, the E-aBNV group secretes CD4+ IFN-γ and IL-2. + The proportion of T cells increased significantly, while the secretion of IL-4 and CD4 increased. + The proportion of T cells did not change significantly, confirming that E-aBNV induces a Th1-biased immune response. CD8 +T cell analysis results showed that the E-aBNV group secreted CD8+ IFN-γ and IL-2. + The proportion of T cells also increased significantly, indicating that antigen-specific cytotoxic T cell responses were effectively induced, enhancing antiviral cellular immune function.

[0033] Example 6: In vivo safety evaluation of E-aBNV (1) Acute toxicity assessment Mice were given a single intramuscular injection of 100 μg E-aBNV. Blood and major organs were collected 12 hours and 42 days later. Hematological and biochemical results are as follows: Figure 23 As shown, compared with the PBS group, the platelet count (PLT), hemoglobin (HGB), red blood cell (RBC), white blood cell (WBC) and their differential counts, as well as liver function indicators (ALT, AST) and kidney function indicators (BUN, CREA) in the E-aBNV group were all within the normal physiological range. The H&E staining results of the major organs (heart, liver, spleen, lung, kidney, and muscle) are shown below. Figure 25 As shown, no obvious tissue structure destruction, abnormal cell morphology, or inflammatory cell infiltration was observed in the E-aBNV group.

[0034] (2) Repeated-dose toxicity assessment Following the immunization schedule (administration on days 0, 14, and 28), blood and major organs were collected on day 42. Hematological and biochemical results are as follows: Figure 23 As shown, all indicators are within the normal range. Weight monitoring results are as follows: Figure 24 As shown, the weight gain trends of mice in each group were consistent, with no significant differences. The H&E staining results of major organs (heart, liver, spleen, lungs, kidneys, and muscles) are as follows. Figure 25 As shown, no obvious tissue structure destruction, abnormal cell morphology, or inflammatory cell infiltration was observed in the E-aBNV group. This indicates that E-aBNV has good biocompatibility.

[0035] In summary, this invention relates to a multi-epitope chikungunya virus vaccine, E-aBNV, based on engineered bacterial nanovesicles, for inducing highly efficient neutralizing antibody responses and Th1-biased cellular immune responses. Following intramuscular injection, E-aBNV activates B cell receptor cross-linking through a high-density antigen array on its surface. Simultaneously, it activates dendritic cells using pathogen-associated molecular patterns carried by E-aBNV itself, promoting antigen presentation and inducing a specific T-cell immune response, thereby preventing chikungunya virus infection. Furthermore, this vaccine exhibits good freeze-drying stability and biosafety.

Claims

1. A multi-epitope fusion protein for preventing chikungunya virus, characterized in that, The fusion protein comprises the E. coli outer membrane anchoring protein ClyA as shown in SEQ ID NO.1, four MHC class I restricted T cell epitopes as shown in SEQ ID NO.2-5, four MHC class II restricted T cell epitopes as shown in SEQ ID NO.6-9, two B cell epitopes as shown in SEQ ID NO.10-11, and the BALB / c mouse immunoglobulin Fc fragment as shown in SEQ ID NO.12, which are tandemly linked by flexible linkers.

2. The multi-epitope fusion protein for preventing chikungunya virus according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

13.

3. The gene encoding the multi-epitope fusion protein for preventing chikungunya virus as described in any one of claims 1 to 2.

4. The gene for preventing chikungunya virus multi-epitope fusion protein according to claim 3, characterized in that, The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO.

14.

5. A recombinant expression vector, characterized in that, The gene comprising the multi-epitope fusion protein for preventing chikungunya virus as described in any one of claims 4 to 5.

6. A genetically engineered bacterium, characterized in that, By converting the recombinant expression vector according to claim 5 to ClearColi BL21 (DE3) Obtained from strains.

7. The use of the multi-epitope fusion protein for preventing chikungunya virus infection as described in any one of claims 1 to 2, the gene as described in claims 3 to 4, the recombinant expression vector as described in claim 5, and the genetically engineered bacteria as described in claim 6 in the preparation of drugs for preventing chikungunya virus infection.

8. A chikungunya virus vaccine, characterized in that, The vaccine comprises engineered nanovesicles obtained by high-pressure homogenization of the genetically engineered bacteria of claim 6.

9. The method for preparing the chikungunya virus vaccine according to claim 8, characterized in that, Includes the following steps: The gene fragment with the nucleotide sequence shown in SEQ ID NO.14 was ligated to the basic vector and then transformed into E. coli. ClearColi BL21(DE3) Competent cells were induced to express IPTG; the bacteria were homogenized by high pressure and purified by differential centrifugation.

10. The preparation method according to claim 9, characterized in that, The high-pressure homogenization conditions were 800 bar and 10 cycles.

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