Flagellum nanofiber vaccine as well as preparation method and application thereof

Abstract

The invention provides a flagellum nanofiber vaccine and a preparation method and application thereof, the vaccine comprises flagellum, a cationic polymer and an antigen, the flagellum is negatively charged, the cationic polymer is self-assembled on the surface of the negatively charged flagellum through electrostatic interaction to form a nanofiber structure, and the antigen is self-assembled on the surface of the negatively charged flagellum. The antigen is coated on the surface of the nanofiber structure. The retention time of the nanofiber vaccine at an injection site is prolonged, and the uptake ability of dendritic cells and macrophages is improved. The immunogenicity of flagellum can significantly promote the maturation of dendritic cells and the polarization of macrophages, and induce the immune response of specific T cells and B cells. In melanoma mice expressing ovalbumin, vaccination with the vaccine carrying ovalbumin not only shows good tolerance, but also shows good inhibition effects on growth and metastasis of tumors in two modes of treatment and prevention.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a flagella nanofiber vaccine and a preparation method and application thereof. Background Art

[0002] Vaccines have become a mainstay of disease prevention and treatment. By coordinating the immune system, vaccines can stimulate effective therapeutic responses and lasting protection without causing severe side effects. Among different types of vaccines, bacteria show great potential because they have innate immunogenicity and can induce strong systemic immune responses. The abundant pathogen-associated molecular patterns on the surface of bacteria and their metabolites such as bacterial toxins can be recognized by immune cells, strongly stimulating the innate immune system and promoting antigen presentation and specific T cell responses. There is evidence that the Bacillus Calmette-Guérin (BCG) vaccine based on the live attenuated strain of Mycobacterium bovis is one of the gold standards for clinical use in the treatment of bladder cancer. Although many bacterial vaccines have entered clinical trials, successful translation is limited by the high risk of infection and associated toxicity. There are reports that even with the approved BCG treatment, patients experience severe side effects such as lymphadenitis, cystitis, and flu-like symptoms. Therefore, infection-related adverse reactions have greatly limited the widespread application of live bacterial vaccines.

[0003] To address the safety issues of vaccines based on live bacteria, bacterial derivatives have been used in vaccine development due to their non-living nature but retaining natural immunogenicity. As typical bacterial derivatives, inactivated bacteria and bacterial ghosts have been widely explored as vaccines in the past decade. The first clinical application of inactivated bacterial vaccines was by Dr. William Coley in the 1890s. Inactivated bacterial vaccines are called Coley toxins. Bacterial ghosts are empty bacterial capsids of Gram-negative bacteria that can be internalized by macrophages and dendritic cells (DCs) to activate T cell immunity. For example, a vaccine produced by loading cancer cell lysates into the bacterial fluid produced by Escherichia coli Nissle 1917 can increase CD8 +T cell circulation, thereby improving the overall survival of lung tumor-bearing animals. Outer membrane vesicles (OMVs) are another important bacterial derivative that is a viable platform for vaccine preparation. OMVs are spherical double-layer nanostructures released by Gram-negative bacteria with many immunogenic similarities inherited from the parent bacteria. Due to their nanosize and inherent immunogenicity, OMVs can be specifically recognized and effectively internalized by immune cells, thereby inducing a strong immune response. Several OMVs-based vaccines have entered clinical trials, demonstrating the advantages of using OMVs to make vaccines. Although the application of inactive derivatives can circumvent the nonspecific distribution of live bacteria in normal tissues, the associated toxicity remains a great challenge due to the retention of complex and undefined bacterial components. For example, the commonly used complete Freund's adjuvant contains dead mycobacteria, which failed in clinical trials due to its severe toxic effects, including local abscesses, chronic granulomas, and ulcerated tissues.

[0004] Compared with live bacteria and their derivatives, single bacterial components have received increasing attention in vaccine development in recent years due to their simple and clear structures. Flagellin, a subunit protein of flagellum, is the most commonly used single immunomodulatory component of bacteria. Flagellin monomers have highly conserved N-terminal and C-terminal domains, which interact in cis to form the core of the filament and the middle hypervariable region, which forms the surface of the filament. A variety of flagellin-based vaccines have been designed with the terminal domains responsible for recognition and signaling of flagellin Toll-like receptor (TLR) 5 and cytoplasmic natural nucleotide-binding and oligomerization domain (NOD)-like receptors, which can induce a strong immune response. It has been documented that vaccination with purified flagellin or tumor cells expressing flagellin can effectively promote tumor regression. However, the application of flagellin-based vaccines faces several difficulties, especially poor stability in immune cells and limited endocytosis related to the inherent hydrophilicity and hydrolyzability of the protein. In addition, the production of flagellin-based vaccines usually requires fusion protein technology, which is time-consuming and expensive. Therefore, alternative forms of vaccines based on single bacterial components that can address these limitations are highly desirable. Summary of the invention

[0005] In view of the defects in the prior art, the purpose of the present invention is to provide a flagella nanofiber vaccine and a preparation method and application thereof.

[0006] According to the first aspect of the present invention, there is provided a flagella nanofiber vaccine, comprising flagella, a cationic polymer and an antigen, wherein the flagella are negatively charged, the cationic polymer self-assembles on the surface of the negatively charged flagella through electrostatic action to form a nanofiber structure, and the antigen is coated on the surface of the nanofiber structure.

[0007] Furthermore, the flagella serve as both a delivery vehicle and an immune adjuvant.

[0008] Optionally, the cationic polymer is PEI. Since PEI is a cationic polymer with positive charge, it can be assembled with negatively charged flagella through electrostatic interaction, and can further bind to negatively charged antigens later.

[0009] Optionally, the cationic polymer is a low-toxic cationic polymer such as chitosan or PMMA.

[0010] Optionally, the antigen is OVA.

[0011] Optionally, the antigen is a viral antigen of S1 protein.

[0012] It should be noted that other negatively charged antigens can also be used to develop different types of flagellar nanofiber vaccines.

[0013] According to a second aspect of the present invention, there is provided a method for preparing the flagella nanofiber vaccine, comprising:

[0014] providing a flagellum, the flagellum being negatively charged;

[0015] Providing a cationic polymer, and self-assembling the cationic polymer on the surface of negatively charged flagella through electrostatic interaction to obtain a nanofiber structure with reversed surface charge;

[0016] The flagellar nanofiber vaccine is obtained by coating antigens on the nanofiber structure with reversed surface charge.

[0017] According to a third aspect of the present invention, there is provided a use of the flagella nanofiber vaccine described above in the preparation of a drug for preventing and / or treating a disease.

[0018] Bacteria-based vaccines have attracted much attention due to their ability to induce a strong systemic immune response. However, the use of live bacteria as therapeutic agents inevitably causes infection-related side effects. The present invention provides a self-adjuvant nanofiber vaccine using flagella from bacteria, where the charge on the flagella surface can be reversed from negative to positive through electrostatic interaction with cationic polymer deposition, which can further mediate the coating of negatively charged antigens on the flagella surface. Taking advantage of the large aspect ratio of flagella, the obtained nanofiber vaccines exhibit a longer retention time at the injection site and enhance cellular internalization of DCs and macrophages. Due to the innate immunogenicity of flagella, these nanofiber vaccines show a self-adjuvant property, strongly promoting DC maturation and macrophage polarization, thereby stimulating strong antigen-specific T cell and B cell immune responses. In a therapeutic setting, immunization of mice carrying melanoma (B16-OVA) overexpressing ovalbumin (OVA) can significantly inhibit tumor growth. At the same time, in the B16-OVA lung metastasis mouse model, antigen-carrying vaccines effectively prevent tumor metastasis in a preventive setting. Notably, these vaccines had negligible toxic side effects on normal tissues. The present invention also demonstrated the versatility of this approach in vaccine development by encapsulating the S1 subunit of the SARS-CoV-2 spike protein. Given these unique features, antigen-coated self-adjuvanted bacterial flagella could provide a simple and flexible platform for the development of a variety of nanovaccines for disease intervention.

[0019] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0020] The present invention uses flagella derived from bacteria and realizes charge reversal through modification with positively charged polymers. The flagella can be combined with negatively charged antigens through electrostatic action to form a coating, thereby constructing a self-adjuvanted nanofiber vaccine based on bacterial flagella. By utilizing the larger aspect ratio of flagella, the retention time of the obtained nanofiber vaccine at the injection site is prolonged, and the uptake capacity of dendritic cells and macrophages is increased. In addition, the immunogenicity of the flagella itself can significantly promote the maturation of dendritic cells and the polarization of macrophages, inducing specific T cell and B cell immune responses. In melanoma mice expressing ovalbumin, immunization with a vaccine carrying ovalbumin not only showed good tolerance, but also showed a better inhibitory effect on tumor growth and metastasis in both treatment and prevention modes. In addition, by coating the SARS-CoV-2 spike protein S1 subunit, the flexibility of the method in vaccine preparation was verified. The present invention can be used to develop various vaccines for use in the preparation of drugs for the prevention and / or treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0022] Figure 1 Schematic diagram of the separation of FLA from Salmonella typhimurium VNP20009 strain;

[0023] Figure 2 In the figure, (a) is a schematic diagram of the preparation method of FLA-OVA, (b) shows the increase of tumor retention and immune cell antigen uptake mediated by FLA-OVA and the related intratumoral immune activation, (c) shows that FLA-OVA with good tolerance inhibits melanoma growth and prevents melanoma lung metastasis;

[0024] Figure 3 The characterization results of FLA-OVA are shown, wherein: (a) is the fluorescence spectra of PBS and FLA-PEI; (b) is the co-localization of FLA bound to rhodamine b and PEI bound to FITC in DC2.4 cells, scale bar: 20 μm; (c) is the Zeta potential of FLA, FLA-PEI and FLA-OVA (n=3); (d) is the AFM image of FLA (scale bar: 300 nm) and FLA-OVA (scale bar: 600 nm); (e) is the average diameter of FLA and FLA-OVA (n=10); (f) is the TEM image of FLA and FLA-OVA, scale bar: 200 nm; (g) is the average diameter of FLA, FLA-PEI and FLA-OVA 1 H NMR spectrum; (h) FTIR spectra of LA, FLA-PEI and FLA-OVA;

[0025] Figure 4 The adsorption rate of OVA on FLA-PEI is shown;

[0026] Figure 5The uptake of FLA-OVA by immune cells is shown, wherein: (a) represents confocal images of DC2.4 cells after incubation with OVA and FLA-OVA for 4 hours, respectively, OVA is bound to FITC, and the scale bar is 50 μm; (b) represents flow cytometric analysis of DC2.4 cells after incubation with PBS, OVA and FLA-OVA for 4 hours; (c) represents the percentage of OVA and FLA-OVA internalized by DC2.4 cells after incubation for 4 hours; (d) represents the MFI of FITC in DC2.4 cells; (e) represents confocal images of Raw 264.7 cells after incubation with FLA-OVA for 4 hours, and the scale bar is 50 μm; (f) represents flow cytometric analysis of Raw 264.7 cells after incubation with PBS, OVA and FLA-OVA for 4 hours; (g) represents the percentage of OVA and FLA-OVA internalized by Raw 264.7 cells after 4 hours of culture; (h) represents the MFI of FITC in Raw264.7 cells;

[0027] Figure 6 The figure shows the uptake of FLA-OVA by DC2.4 cells, where FLA is labeled with rhodamine b and OVA is labeled with FITC; (a) Flow cytometric analysis of FLA-OVA uptake by DC2.4 cells after 4 hours of culture. (b) FLA-OVA uptake by DC2.4 cells after different treatments + and (c)OVA + Cell percentage; data are expressed as mean ± SD (n = 4); statistical analysis was performed using one-way ANOVA and LSD post hoc test; ***p < 0.001;

[0028] Figure 7 Figure 2 shows the uptake of FLA and FLA-PEI by DC2.4 cells; DC2.4 cells were incubated with FLA or FLA-PEI for 4 hours, and FLA was labeled with rhodamine b; (a) is the flow cytometric analysis of FLA and FLA-PEI; (b) is the flow cytometric analysis of FLA + Cell percentage; Tukey's method was used to identify and delete outliers in SPSS; data are expressed as mean ± SD (n = 6); Student's t test was used for statistical analysis; ns, no significant difference;

[0029] Figure 8The uptake of FLA-OVA by DCs is shown; FLA and OVA were labeled with rhodamine b and FITC, respectively; flow cytometric analysis of BMDCs after treatment with FLA, OVA and FLA-OVA for (a) 2, (c) 12 and (e) 24 hours, respectively; the percentage of BMDCs after treatment with FLA, OVA and FLA-OVA for (b) 2, (d) 12 and (f) 24 hours, respectively; data are expressed as mean ± SD (n = 3-4); statistical analysis was performed using one-way ANOVA and LSD post hoc; *p < 0.05, ***p < 0.001;

[0030] Fig. 9 Shown are the effects of FLA-OVA on DC activation and macrophage polarization in vitro; (a) CD80 + , (c)CD86 + , (e) SIINFEKL-H-2Kb + Flow cytometry analysis of cells after different treatments; (b) CD80 + (d) CD86 + and (f) SIINFEKL-H-2Kb + Percentage of BMDCs; Representative flow cytometric analysis of (g) M1 and (i) M2 after different treatments; Percentage of (h) M1 and (j) M2 after different treatments; (k) CD80 + CD86 + BMDCs and (l)SIINFEKL-H-2Kb + Percentage of BMDCs; (m) M1 / M2 ratio in different treatment groups; SPSS was used to identify and delete outliers using Tukey's method; Data are expressed as mean±SD (n=3-6); Statistical analysis was performed using one-way ANOVA and LSD post hoc; *p<0.05, **p<0.01, ***p<0.001; ns, no significant difference;

[0031] Fig.10 Shows the activation effect of FLA-OVA on DCs in vitro; (a) MHC-II + Flow cytometric analysis of BMDCs; (b) MHC-II after different treatments + Percentage of BMDCs; data are expressed as mean ± SD (n = 3); statistical analysis was performed using one-way ANOVA and LSD post hoc; *p < 0.05;

[0032] Fig.11 Figure 3 shows the mechanism of FLA-OVA-mediated immune response; (a) CD80 expression in the presence or absence of TLR5 inhibitor TH1020. + CD86 +BMDCs, (b) SIINFEKL-H-2Kb + BMDCs, (c) representative flow cytometric analysis of M1 / M2;

[0033] Fig.12 The retention of FLA-OVA in vivo is shown, wherein: (a) mice treated with PBS, OVA (FITC-labeled) or FLA-OVA (FITC-labeled OVA) at predetermined time intervals were imaged and (b) quantitatively analyzed by an in vivo imaging system; (c) quantitative analysis and (d) in vivo imaging system images of sliced ​​tissue at the injection site 24 hours after injection; (e) injection site and (f) fluorescence intensity of FITC in the collected blood sample detected by a fluorescence spectrophotometer;

[0034] Fig.13 Figure 2 shows the in vivo immune response induced by FLA-OVA; (a) mouse immunization experiment design; C57BL / 6 mice were subcutaneously injected with PBS, OVA, FLA and FLA-OVA on days 0, 7 and 14, respectively; on day 21, the mice were euthanized, and the injection site tissues, inguinal lymph nodes and spleen samples were collected to evaluate the immune response; (b) CD11b + cells, (c)CD11c + Cells and (d) CD3 + The percentage of cells at the injection site; (e) M1 / M2 and (f) M2 percentage in nodes; (g) M1 / M2 in spleen; CD80 + CD86 + The percentage of DC in (h) inguinal lymph nodes and (i) spleen; (j) SIINFEKL-H-2Kb + DC and (k)CD8 + The percentage of T cells in the inguinal lymph nodes; (l) CD8 + With CD4 + Ratio of T cells; (m) IFN-γ + CD8 + T cell percentage and (n) IFN-γ + CD4 + T cell percentage; OVA + CD8 + The percentages of T cells in the inguinal lymph nodes and spleen are (o) and (p), respectively; (q) Ki67 in the spleen + CD8 + The percentage of T cells; Tukey's method was used to identify and delete outliers in SPSS; data are expressed as mean ± SD (n = 6-7); statistical analysis was performed using one-way ANOVA and LSD; *p < 0.05; **p < 0.01; ***p < 0.001;

[0035] Fig.14 Shown is the dose escalation response of FLA-OVA in vivo; plasma OVA-specific (a) IgG1 and (b) IgG levels; and (c) SIINFEKL-H-2Kb in spleen. + DC and (d) OVA + CD8 + Percentage of T cells; (e) Spleen weight; (f) Tumor growth after PBS and increasing doses of FLA-OVA treatment; (g) Image of B16-OVA tumor specimens on day 10 after treatment; Scale bar: 10 mm; Outliers were identified and deleted using Tukey's method in SPSS; Data are expressed as mean ± SD (n = 6); Statistical analysis was performed using one-way or two-way ANOVA and LSD post hoc; *p < 0.05; **p < 0.01; ***p < 0.001; n, no significant difference;

[0036] Fig.15 Shows the in vivo tumorigenicity and stability of B16-OVA cells; (a) tumor growth curve; (b) digital image of B16-OVA tumors collected on day 13 after inoculation; n = 5; scale bar: 10 mm;

[0037] Fig.16 The anti-tumor effect of FLA-OVA in the B16-OVA mouse model is shown, where: (a) Immunofluorescence staining of tumor tissues after different treatments, red, green, and blue represent the signals of CD4, CD8, and cell nucleus, respectively; scale bar: 50 μm; (b) Tumor growth after different treatments; (c) Representative images of B16-OVA tumors 10 days after treatment; scale bar: 10 mm; (d) Individual tumor growth curves of PBS, (e) OVA, (f) FLA, and (g) FLA-OVA; (h) H&E staining of tumor tissues; scale bar: 50 μm;

[0038] Fig.17 The effect of FLA-OVA on IFN-γ in vivo + CD4 + T cell activation; (a) Experimental design of immune activation in B16-OVA tumor-bearing mice; C57BL / 6 mice were inoculated with B16-OVA on day 7; when the tumor volume reached ~100mm 3 B16-OVA tumor-bearing mice were intratumorally injected with PBS, OVA, FLA, and FLA-OVA, respectively. After treatment, tumors and tumor-draining lymph nodes were sampled to evaluate immune responses. (b) Flow cytometric analysis and (c) expression of IFN-γ in tumors + CD4 +T cell profiles; (d) flow cytometric analysis and (e) inguinal lymph node IFN-γ + CD4 + T cell contour map; data are expressed as mean ± SD (n = 4); statistical analysis was performed using one-way ANOVA and LSD post hoc; *p < 0.05; **p < 0.01;

[0039] Fig.18 The effect of FLA-OVA on IFN-γ in vivo + CD8 + T cells and OVA + T cell activation; (a) tumor and (b) inguinal lymph node IFN-γ + CD8 + Flow cytometric analysis of T cells; (c) OVA in tumors + CD8 + Flow cytometry analysis of T cells; data are expressed as mean ± SD (n = 4); statistical analysis was performed using one-way ANOVA and LSD post hoc; **p < 0.01, **p < 0.001;

[0040] Fig.19 Shows the experimental design for in vivo treatment of B16-OVA tumor-bearing mice;

[0041] Fig. 20 The fluctuation of mouse body weight during treatment is shown; the data are expressed as mean±SD (n=8);

[0042] Fig.21 The biosafety of FLA-OVA is shown; H&E staining images of the heart, liver, spleen, lung, and kidney tissues of B16-F10 tumor-bearing mice treated with PBS, OVA, FLA, and FLA-OVA, respectively; scale bar: 100 μm;

[0043] Fig. 22The preventive effect of FLA-OVA on lung metastasis in B16-OVA mice is shown, wherein: a) Experimental design to evaluate the preventive effect of lung metastasis after treatment with PBS, OVA, FLA and FLA-OVA; mice were subcutaneously administered on days 21, 14 and 7, respectively, and intravenously injected with B16-OVA tumor cells on day 0. Mice were euthanized, and lung and spleen tissues were collected on day 14; b) Digital photos of lungs collected from treated mice; c) The number of metastatic nodules in digital images of lungs; when the number of lung metastases exceeded 150, the number of nodules was estimated by area calculation; d) H&E staining of lung tissues from treated mice; dotted circles are lung tumor metastases; scale bar: 500 μm; e) The number of metastatic nodules in H&E images of lungs; f) Spleen weight; Data are expressed as mean ± SD (n = 3-9); One-way ANOVA and LSD were used for statistical analysis; *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference;

[0044] Fig.23 Pie chart showing melanoma lung metastasis;

[0045] Fig.24 The anti-tumor effects of FLA+OVA and FLA-OVA in the B16-OVA mouse model are shown; tumors were collected after 10 days of treatment; (a) Tumor growth curves under different treatments; (b) Representative digital images of B16-OVA tumors, scale bar: 10 mm; (c) H&E staining of tumor tissue sections, scale bar: 50 μm; (d) Immunofluorescence staining of tumor tissue sections, red, green, and blue are the signals of CD3, CD8, and cell nucleus, respectively, scale bar: 50 μm; data are expressed as mean ± SD (n = 8); statistical analysis was performed using one-way ANOVA or two-way ANOVA and LSD post hoc; *p < 0.05;

[0046] Fig.25 The preventive effects of FLA-OVA and FLA+OVA in the lung metastasis model are shown; (a) Digital images of mouse lungs after different treatments; (b) Tumor lung metastasis rate, when lung metastasis occurs, the metastasis rate is recorded as 1, otherwise, the metastasis rate is recorded as 0; (c) Spleen weight; Data are expressed as mean±SD (n=6-8), and statistical analysis was performed using one-way ANOVA and LSD post hoc, *p<0.05, ***p<0.001;

[0047] Fig.26Figure 4 shows the activation effect of S1-adsorbed FLA (FLA-S1) on B cells and T cells, where: (a) preparation of FLA-S1; (b) schematic diagram of experimental design; to evaluate the versatility of the FLA-based nanofiber vaccine platform, healthy C57BL / 6 mice were subcutaneously injected with PBS, Spike 1 (S1), FLA and FLA-S1 on days 0 and 14, respectively; the mice were then euthanized and spleen tissues were harvested on day 21 to evaluate cellular immune activation responses; immunofluorescence assay of spleen (c) B220 and (d) GL7; B220 (red), GL7 (green) and DAPI (nucleus); scale bar: 20 μm; flow cytometry assay of (e) CD69 + The percentage of B cells and (f) S1-specific B cells; (g) CD3 + cells, (h)S1-specific CD3 + cells, (i) S1-specific CD4 + T cells and (j) S1-specific CD8 + T cell percentage; SPSS was used to identify and delete outliers using Tukey's method, data are expressed as mean ± SD (n = 6-7), and statistical analysis was performed using one-way ANOVA and LSD, *p < 0.05, **p < 0.01, ***p < 0.001;

[0048] Fig. 27 Characterization of FLA-S1 is shown, (a) Zeta potential of FLA, FLA-PEI, and FLA-S1 (n=5); outliers were identified and deleted using Tukey's method through SPSS; (b) TEM images of FLA and FLA-S1; scale bar: 200 nm; (c) average diameters of FLA and FLA-s1 (n=10), statistical analysis was performed using Student's t test, ***p<0.001. DETAILED DESCRIPTION

[0049] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0050] The materials involved in the following embodiments are as follows:

[0051] Luria-Bertani (LB) liquid medium and PBS were purchased from Sangon Biotech. Hydrochloric acid (HCl) was purchased from Sinopharm Chemical Reagent Co., Ltd. Ammonium sulfate and PEI were obtained from Adamas. Dialysis bags were provided by Shanghai Green Bird Technology Development Co., Ltd. Bicinchoninic acid (BCA) protein assay kit was purchased from Tiangen. RPMI 1640 medium, DMEM medium, trypsin, and fetal bovine serum (FBS) were purchased from Gibco. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) were obtained from Peprotech. FITC-conjugated OVA (OVA-FITC) was purchased from Solarbio. FITC-conjugated CD11c antibody, PE-conjugated CD80 antibody, APC-conjugated CD86 antibody, PE-cy7-conjugated CD206 antibody, PE-conjugated major histocompatibility complex class II (MHC-II) antibody, APC-conjugated SIINFEKL-H-2Kb antibody, superphospho-cy5.5-conjugated CD3 antibody, superphospho-cy5.5-conjugated CD11b antibody, FITC-conjugated CD4 antibody, superphospho-cy5.5-conjugated CD4 antibody, PE-conjugated CD8 antibody, PE-cy7-conjugated interferon γ (IFN-γ) antibody, APC-cy7-conjugated CD3 antibody, APC-conjugated TET antibody, PE-conjugated F4 / 80 antibody, FITC-conjugated CD206 antibody, and FITC-conjugated Ki67 antibody were purchased from Biolegend. HRP-conjugated goat anti-mouse IgG or IgG1 was purchased from Abcam. 6- to 8-week-old C57BL / 6 mice, weighing approximately 19 g, were purchased from Beijing Veterinary Laboratory Animal Technology Co., Ltd. All animal experiments were performed in accordance with the guidelines evaluated and approved by the Ethics Committee of the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (A2023017).

[0052] Statistical analysis: All values ​​in the following examples are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS statistical software version 24, and multiple comparisons between groups were determined using LSD post hoc or Student's t test. ***P<0.001, **P<0.01, *P<0.05 were considered statistically significant.

[0053] The term FITC and fitc have the same meaning, and rhodamine b and rhodamine B have the same meaning.

[0054] Example 1

[0055] Preparation and characterization of flagellar nanofiber vaccines

[0056] Isolation of FLA from attenuated Salmonella typhimurium: Reference Figure 1, 20 μL of attenuated VNP20009 solution was added to 2 mL of LB liquid culture medium (VNP20009 was inoculated in Luria-Bertani (LB) liquid culture medium at 1:100), and pre-cultured at 37°C with shaking (200 rpm) for 4 hours. Next, 10 mL of the pre-culture suspension was added to 1 L of fresh LB culture medium and shaken (200 rpm) at 37°C overnight. Salmonella cells were centrifuged at 5000 g for 30 minutes and then suspended in a PBS solution. FLA was isolated from bacteria according to the description of Ibrahim et al. (MLKalmokoff, KFJarrell, SFKoval, J.Bacteriol.1988,170,1752.). Then adjust to pH 2.0 with 1 M HCl and continue stirring at room temperature for 30 minutes. After FLA separation, the bacterial cells were separated by centrifugation at 5000 g for 30 minutes. Ultracentrifuge at 100,000 g for 1 hour at 4°C to separate insoluble substances at pH 2.0. Slowly add ammonium sulfate to adjust the supernatant pH to 7.2, and stir vigorously until the ammonium sulfate concentration reaches 2.67m. After stirring for 30 minutes, incubate at 4°C overnight, and centrifuge at 21130g for 15 minutes at 4°C. After salinization, dissolve the residue in distilled water and transfer to a dialysis bag (50kDa MWCO). Dialysis is performed in excess distilled water at 4°C, and the water is changed every 6 hours. After 2 days of dialysis, the pure FLA is freeze-dried and stored at -80°C for later use.

[0057] Preparation and characterization of FLA-PEI and FLA-OVA: Reference Figure 2 In a, the purified FLA was lyophilized and dispersed in PBS, and 25 μL of the above FLA solution was reacted with BCA reagent. The optical density (OD) value was measured at 562 nm, and the FLA concentration was calculated using the bovine serum albumin (BSA) standard curve. In order to form FLA-PEI, PEI dissolved in PBS was slowly dripped into the shaking FLA solution, vortexed for 10 minutes, washed with PBS, and centrifuged at 10000g for 5 minutes with a 50kDa ultrafiltration tube to remove unbound PEI, thereby preventing the subsequent assembly of OVA. Then the OVA solution was slowly dripped into the FLA-PEI and vortexed for 10 minutes to obtain FLA-OVA. The mass ratio of each component is FLA:PEI:OVA=1:1:3.

[0058] Since PEI has certain cytotoxicity, the PEI used in the preparation process is a relatively low dose. The electrostatic interaction between FLA and PEI allows for full binding under the condition of vortexing for 10 minutes. The amount (mass) of OVA does not exceed 10 times that of FLA.

[0059] The self-adjuvanted nanofiber vaccine was prepared by coating cationic polyethyleneimine (PEI) and OVA model antigen on Salmonella-derived flagella through layer-by-layer self-assembly of nanotechnology. The flagella obtained from VNP20009 (called FLA) were separated and purified from the culture medium through a series of procedures including acidification, salting out and ultracentrifugation. The positively charged PEI was simply mixed with the negatively charged FLA and deposited on the flagellar surface through electrostatic interaction. After removing the excess PEI by ultrafiltration centrifugation, the positively charged PEI-coated FLA (defined as FLA-PEI) was further mixed with the negatively charged OVA, which was easily coated on the surface to form a nanofiber vaccine (defined as FLA-OVA).

[0060] To estimate the modification of PEI on flagella, PEI was labeled with fluorescein isothiocyanate (FITC). After purification, FLA-PEI was scanned with a microplate reader to detect FITC fluorescence. FLA and rhodamine b isothiocyanate were dissolved in 0.1M sodium carbonate-sodium bicarbonate buffer (pH 9.2) and reacted at room temperature for 24 hours. After dialysis with a MWCO 50KD dialysis bag for 24 hours, purified rhodamine b-labeled FLA was obtained. In order to evaluate the colocalization of rhodamine b-labeled FLA and FITC-labeled PEI, DC2.4 cells were used to internalize FLA-PEI. Next, nuclear magnetic resonance and infrared spectroscopy were used to further characterize the modification of FLA in the FLA-OVA nanovaccine by PEI. FLA, FLA-PEI and FLA-OVA were dissolved in deuterium oxide (D2O), and 1H NMR experiments were performed on a Bruker AVANCE III HD 400 spectrometer. FLA, FLA-PEI and FLA-OVA were freeze-dried into powders, and the samples were subjected to FTIR experiments at 298K on a Thermo Fisher Nicolet 6700 spectrometer. The morphology of FLA and FLA-OVA was characterized by 120KV TEM (Thermo Fisher, Tecnai G2spirit Biotwin) and AFM (Oxford Instruments, MFP-3D). The diameters of FLA and FLA-OVA were obtained by randomly measuring 10 nanofibers in the TEM images using Nano Measure software and statistically analyzed. The Zeta potential of FLA, FLA-PEI and FLA-OVA was measured using a nanoparticle size Zeta potentiometer (Brookhaven Instruments Corporation, omni). To determine the loading efficiency of the antigen, OVA solution was added to the FLA-PEI nanofibers to increase the ratio of antigen (from FLA:PEI:OVA=1:1:1 to FLA:PEI:OVA=1:1:100). After 10 minutes of constant vortexing, the unbound OVA was removed by centrifugation at 10,000 g for 5 minutes using a 50 kDa ultrafiltration tube to obtain FLA-OVA nanofibers. The permeate containing unbound OVA was collected and the concentration of OVA was quantified using a BCA assay kit. The loading efficiency was calculated as the total amount of OVA minus the unbound portion.

[0061] Reference Figure 3 , PEI was first labeled with fluorescein isothiocyanate (FITC) to confirm the effective deposition of PEI on FLA. Figure 3As shown in Figure a, a fluorescence emission peak of about 519 nm was observed on FLA-PEI, indicating the appearance of PEI on FLA. The colocalization of FITC-labeled PEI and rhodamine B-labeled FLA further proved the formation of FLA-PEI ( Figure 3 Compared with FLA, the Zeta potential of FLA-PEI increased from -12.4 ± 4.4 mV to -3.7 ± 1.5 mV (Fig. Figure 3 (c). With the deposition of OVA on the FLA surface, the Zeta potential dropped to -13.5 ± 2.5 mV. Atomic force microscopy (AFM) images showed that the FLA surface coated with OVA had obvious nanostructures, with a diameter increased by 2 times ( Figure 3 (d) Transmission electron microscopy (TEM) further confirmed the preparation of FLA-OVA. Figure 3 As shown in Figures e and f, the diameter of unmodified FLA is 25 nm, and after OVA is fixed on the surface of FLA, the diameter increases to 39 nm, forming a layer-by-layer nanofiber structure. 1 H NMR) spectrum found that PEI has characteristic peaks between 2.66 and 2.88 ppm, indicating that PEI is deposited on the surface ( Figure 3 g). Compared with FLA, the Fourier transform infrared (FTIR) spectrum of FLA-OVA showed a significant difference at 2850 and 2920 cm -1 The additional bands are attributed to the vibration of -CH2 groups in PEI ( Figure 3 h). When the OVA dosage is less than 100 μg, refer to Figure 4 , quantitative analysis showed that the absorption rate of OVA by PEI-modified FLA was ~99%. Taken together, these results indicate that bacterial flagellar nanofiber vaccines were successfully prepared by layer-by-layer self-assembly.

[0062] Example 2

[0063] Role of FLA in antigen uptake in vitro

[0064] The uptake of exogenous antigens by DC is crucial for the subsequent antigen presentation of major histocompatibility complex (MHC) molecules and the activation of T cells, ultimately triggering an antigen-specific immune response. Due to the large aspect ratio, nanofibers can effectively enter cells. The present invention embodiment studies the efficiency of FLA-mediated antigen uptake by directly incubating DC2.4 cells. For ease of observation, OVA was labeled with FITC. DC2.4 cells were incubated with phosphate-buffered saline (PBS), OVA (30 μg mL -1) and FLA-OVA (equal amount of OVA) were incubated at 37°C for 4 h. Specifically, the cellular uptake experimental process of FLA-OVA was as follows: BMDCs were extracted from 6-week-old female C57BL / 6 mice (femur and tibia), and 10% bovine serum, 100 μg mL -1 Penicillin, 100 μg mL -1 Streptomycin and 20 ng mL -1 GM-CSF, culture for 7 days. The medium was changed every 3 days. The DCs were collected by centrifugation on day 8. Cellular uptake of FLA, OVA and FLA-OVA was measured using BMDCs, DC2.4 cells and Raw 264.7 cells, respectively. 3 × 105 BMDCs were cultured in 24-well plates. FLA (5 μg mL -1 , coupled with rhodamine b), OVA (30 μg mL -1 , coupled with FITC), FLA-OVA (FLA, 5 μg mL -1 ;PEI, 5μg mL -1 ; Add OVA, 30 μg mL -1 ) and incubated for 4 h. Cells were collected from 24-well plates, washed three times, and analyzed by flow cytometry. DC2.4 cells and Raw 264.7 cells were analyzed for uptake as above. Five 10 × 4 DC2.4 cells or Raw 264.7 cells were seeded into confocal dishes and allowed to adhere overnight to analyze FLA-OVA intracellular trafficking. Cell culture medium was replaced with FLA (5 μg mL -1 )、OVA(30μg mL -1 Combined with FITC) and FLA-OVA (FLA, 5 μg mL -1 ; OVA, 30 μg mL -1 ). Incubate at 37°C for 4 h. Then discard the supernatant, wash the adherent cells 3 times with PBS, and add 5 μg mL -1 The cells were incubated with Hoechst 33342 fluorescent dye for 15 min in the dark at room temperature, washed three times with PBS, and imaged under a laser scanning confocal microscope (Leica TCS SP8).

[0065] Confocal images of cells incubated with FLA-OVA showed clear OVA signals compared with free OVA ( Figure 5 (a) Flow cytometry was used to quantitatively detect the percentage of DC2.4 cells internalizing OVA. Figure 5As shown in Figures b and c, after loading OVA on the FLA surface, the percentage of cells internalizing OVA increased from ~5% to ~45%. In addition, the mean fluorescence intensity (MFI) of cells cultured with FLA-OVA increased by 7 times compared with cells cultured with free OVA ( Figure 5 d), which is in good agreement with the confocal imaging results. To verify the enhanced cellular uptake mediated by FLA, it was observed that FLA was internalized by binding to rhodamine b. Figure 6 As shown in a and b, about 60% of DC2.4 cells showed intracellular FLA signals. This also shows that OVA coating does not affect the absorption of FLA. Due to the advantage of FLA in cellular uptake, the internalized OVA increased by about 10 times compared with free OVA ( Figure 6 c). In order to clarify whether the enhanced uptake is due to the positive charge of PEI or the morphological aspects of FLA, the present invention incubated cells with FLA and FLA-PEI respectively. Here, FLA was labeled with rhodamine b. Figure 7 As shown, the deposition of PEI on the surface did not enhance the cellular uptake of FLA. The improvement of FLA-mediated oocyte uptake is closely related to the nanofibrous structure of FLA. The embodiment of the present invention also measured the uptake of FLA-OVA by using bone marrow-derived dendritic cells (BMDC). The OVA-specific BMDCs activation and maturation experimental process is as follows: BMDCs were collected and dispersed in a 24-well plate at a density of 3×105 cells per well, and then co-cultured with FLA, OVA and FLA-OVA in RPMI 1640 medium at 37°C for 24 hours. DCs were collected and incubated with anti-CD11c-FITC, anti-MHCII-PE, anti-CD80-PE, anti-CD86-APC, and anti-SIINFEKL-H-2Kb-APC antibodies at room temperature for 45 minutes, washed twice with PBS, and detected by flow cytometry (BD LSRFortessa). By using PBS, OVA (30 μg mL -1 )、FLA(5μg mL -1 ) and FLA-OVA (FLA and OVA are equivalent) were incubated with mouse BMDCs for 2, 12 and 24 hours. + The cell ratio was significantly higher than that of the free OVA group. Figure 8 As shown in Figures a and b, only ~3% of cells internalized OVA after incubation with free antigen. In contrast, this proportion increased to ~10% after incubation with FLA-OVA. As the incubation time was extended to 12 and 24 h, OVA in the FLA-OVA group increased. + The proportion of cells increased to 40%. However, there was almost no change in the free OVA group ( Figure 8These results suggest that the nanofibrous structure of FLA can largely enhance the uptake of antigens by DCs, which is a prerequisite for the initiation of antigen-specific immunity.

[0066] In order to study whether other types of immune cells also have higher cellular uptake mediated by FLA-OVA, macrophages were selected in the present embodiment, considering that macrophages have the ability to present antigens. The macrophage polarization experiment process is as follows: M2 macrophages were polarized from Raw 264.7 cells. Briefly, 1×106 264.7 cells were incubated in the presence of IL-4 (20 ng mL -1 ) in RPMI 1640 medium in 12-well plates for 24 hours to form M2 macrophages. The obtained M2 macrophages were incubated with FLA (5 μg mL -1 )、OVA(30μg mL -1 ), FLA-OVA (FLA, 5 μg mL -1 ) incubated; PEI (5 μg mL -1 ); OVA (30 μg mL -1 ) and injected for 4 hours. After incubation with APC-coupled CD86 (1:200) for 30 minutes, the cells were treated with fixative and permeabilization solutions. Then, PE-Cy7-coupled CD206 (1:200) was incubated with the cells for 40 minutes. Flow cytometry was used to detect macrophages M1 and M2. -1 ) and FLA-OVA (equal amount of OVA) were incubated in Raw 264.7 cells to observe the uptake of OVA by cells. Similar to DCs, after 4 hours of incubation, the OVA fluorescence signal in Raw 264.7 cells in the FLA-OVA group was stronger, as shown in Figure 2. Figure 5 Compared with free OVA culture, the proportion of macrophages internalizing OVA was approximately 70 times higher ( Figure 5 f, g). As the uptake of OVA increases, + The MFI of Raw 264.7 cells was approximately 10 times higher than that of the free OVA group ( Figure 5 (h) Overall, the nanofibrous structure-assisted cellular internalization can effectively promote the uptake of antigens by professional antigen-presenting cells.

[0067] Example 3

[0068] Activation of immune cells by FLA-OVA in vitro

[0069] The experimental process of the mechanism of anti-tumor immune activation mediated by FLA-OVA is as follows: In order to clarify the mechanism of FLA-OVA activation of DCs and upregulation of antigen presentation and polarization of macrophages to M1 phenotype, DCs or macrophages were treated with PBS, OVA, FLA and FLA-OVA respectively. The subsequent experimental process can refer to the process in the above embodiment.

[0070] After antigen uptake, the subsequent effective activation of immune cells is crucial for initiating an immune response. To verify the activation effect of FLA-OVA on DCs, BMDCs were incubated with PBS, LPS, OVA (30 μg mL -1 )、FLA(5μg mL -1 ) and FLA-OVA (FLA and OVA are equivalent) were incubated for 24 hours. Then the expression levels of CD80, CD86, and MHC-II were detected by flow cytometry. BMDCs stimulated with FLA and FLA-OVA showed the highest upregulation of CD80, CD86, and MHC-II co-stimulatory molecules ( Fig. 9 In ad and Fig.10 ), even stronger than the classic toll-like receptor 4 agonist LPS. The excellent effect of FLA in activating DCs proves the self-adjuvant properties of FLA-OVA. Next, it will be studied whether the maturation of DCs can promote antigen presentation. BMDCs were also incubated with PBS, OVA, FLA and FLA-OVA for 24 hours. Flow cytometry was used to detect the expression level of SIINFEKL-H-2Kb. Free OVA and FLA are almost ineffective for antigen presentation ( Fig. 9 e, f). The expression of OVA-specific MHC-I complexes on the surface of DCs was significantly increased after FLA-OVA stimulation compared with all control groups, indicating that FLA-OVA is particularly effective in promoting antigen presentation by DCs. These data suggest that enhanced antigen uptake and the self-adjuvant properties of FLA-OVA can promote DC maturation and subsequent antigen presentation.

[0071] In addition to dendritic cells, macrophages, as another important member of professional antigen-presenting cells, are one of the key first-line defenses against pathogens. M2-like macrophages play a key role in phagocytosis of apoptotic cells, anti-inflammation, and carcinogenesis. In contrast, M1 macrophages cause inflammation, destroy tissue integrity, and inhibit tumor progression. The polarization of macrophages is important for maintaining immune homeostasis. In particular, the repolarization of tumor-associated macrophages from the anti-inflammatory M2 phenotype to the pro-inflammatory M1 phenotype can reverse the tumor immunosuppressive microenvironment. -1 ) and FLA-OVA (equal amount of OVA) were incubated with Raw 264.7 cells for 4 hours to observe the effect of FLA-OVA on macrophage polarization. Fig. 9As shown in Figures g and j, among all treatment groups, FLA-OVA most effectively triggered the repolarization of macrophages from M2 to M1 phenotype, which was supported by the expression of CD86 + The proportion of cells increased significantly and CD206 + The regulatory effect of FLA-OVA on macrophage polarization can be attributed to the innate immunogenicity of FLA. Flagellin is a key adjuvant of FLA. It has been widely demonstrated that flagellin monomers have highly conserved N-terminal and C-terminal domains. The terminal domains are responsible for the recognition and signaling of Toll-like receptor (TLR) 5, which subsequently triggers a strong immune response. TH1020 is believed to inhibit downstream signal transduction mediated by TLR5 / flagellin complex formation. To elucidate the mechanism of FLA-mediated immune response, PBS, FLA (5 μg mL -1 ) and FLA-OVAFLA, 5 μg mL -1 ) and stimulated BMDCs treated with or without TH1020; OVA (30 μg mL -1 ) for 24 hours. The effects of FLA and FLA-OVA on immune cell activation and antigen presentation were abolished by TH1020 ( Fig. 9 k, 1 and Fig.11 In addition, the effects of FLA and FLA-OVA on macrophage polarization were also inhibited by TH1020 ( Fig. 9 Chinese Fig.11 (c) These findings demonstrate that FLA-OVA can modulate the activation and response of DCs and macrophages. The enhanced immunomodulatory effect of FLA-OVA may be related to the activation of TLR5 by FLA.

[0072] Example 4

[0073] Retention and immune activation effects of FLA-OVA in vivo

[0074] The experimental process of in vivo immune cell activation is as follows: In order to study the immune response induced by FLA-OVA, the present invention embodiment conducted experiments in tumor-bearing mice. 6-week-old C57BL / 6 mice were inoculated with 1×106 B16-F10 cells and the tumor volume reached 100mm. 3 After that, FLA-OVA was injected intratumorally once every other day for a total of 4 times. The mice were euthanized at the end of the treatment. Tumor-draining lymph nodes and tumor tissues were dissected from vaccinated mice, mechanically disrupted, and then centrifuged at 1000g for 5 minutes. The samples were then filtered through a 200-mesh nylon mesh filter to obtain a single-cell suspension, which was collected and washed three times with PBS. To detect CD8 +T cells, anti-CD3-APC and anti-CD8-PE were incubated with approximately 2×106 cells at room temperature for 30 minutes, washed twice with PBS to remove unbound antibodies, and detected on a flow cytometer. Anti-CD3-APC and anti-CD4-FITC were incubated with tissue cells as above to measure CD4 + T cell percentage. To analyze the effect of FLA-OVA on tumor and lymph node cytotoxic T cells, the collected tissue cells were incubated with anti-CD3-APC, anti-CD8-PE, and anti-IFNγ-PE-Cy7. To detect Th1 cells, anti-CD3-APC, anti-CD4-PE, and anti-IFNγ-PE-Cy7 were incubated with the above cells. The antibody dilution ratio was 1:200. The antibody-incubated cells were analyzed by flow cytometry.

[0075] The experimental process of antigen retention and blood distribution at the injection site was as follows: 6-week-old male C57BL / 6 wild-type mice were divided into 4 groups and subcutaneously injected with PBS, free FITC-bound OVA (300 μg / mouse) and FLA-OVA (equivalent OVA, 100 μg / mouse FLA). The injected mice were imaged by an in vivo imaging system (IVIS 6Lumina II, Caliper) at predetermined time intervals to evaluate the retention of OVA at the injection site. The fluorescence intensity of the injected mice was analyzed by Live Image 4.2. To further evaluate the fluorescence intensity and blood distribution at the injection site, C57BL / 6 mice were euthanized 24 hours after administration, and tissues and blood at the injection site were collected. The sampled tissues at the injection site were immersed in PBS and homogenized in the dark. The blood was centrifuged at 2000g for 10 minutes to obtain plasma. OVA-FITC in the homogenate and plasma was detected by a fluorescence spectrophotometer.

[0076] The in vivo retention and biodistribution of vaccines after injection are important factors affecting their safety and efficacy. Due to the large aspect ratio, nanofiber structures show long-term retention in vivo compared to small molecules, macromolecular biologics, and even nanoparticles. To test this property, the present embodiment evaluated the in situ retention of FLA-OVA in mice after subcutaneous injection. Both PBS and free FITC-labeled OVA were used as controls. Fig.12As shown in a and b, compared with free OVA, the retention time of FLA-OVA at the injection site is significantly prolonged, which can be reflected by the retention of the fluorescence signal monitored by the in vivo imaging system. Almost no fluorescence was detected 24 hours after the injection of free OVA, while the fluorescence signal remained strong after the administration of FLA-OVA. The diffusion of the labeled vaccine may be the reason for the enhanced fluorescence 4 or 6 hours after subcutaneous injection. The embodiment of the present invention further measures the fluorescence intensity of the sliced ​​tissue around the injection site. In the in vivo imaging system images, the tissue injected with FLA-OVA showed obvious fluorescence, while this fluorescence was not observed in the PBS and free OVA groups ( Fig.12 In vivo imaging and fluorescence spectrophotometer measurements showed similar increases in fluorescence intensity, which were quantified as far exceeding the control group ( Fig.12 It is worth noting that the retention time of FLA-OVA in the blood was significantly prolonged compared with PBS and free OVA ( Fig.12 f), which may stimulate an effective systemic immune response.

[0077] To evaluate the immunomodulatory effects of FLA-OVA in vivo, mice were subcutaneously injected with PBS, OVA (30 μg per mouse), FLA (10 μg per mouse), and FLA-OVA (FLA, 10 μg per mouse); OVA (30 μg / mouse) on days 0, 7, and 14 ( Fig.13 (a). The spleen, inguinal lymph nodes, and tissues surrounding the injection site were then collected on day 21 to assess the immune response elicited. Encouragingly, the proportion of macrophages, DCs, and T cells at the injection site was significantly increased in mice given FLA-OVA compared with all control groups ( Fig.13 The increase in immune cell infiltration at the injection site further promoted the increase and activation of immune cells in the draining lymph nodes and spleen, which is consistent with the results of previous studies. Fig.13 As shown in the figure, FLA-OVA induced M1 polarization and reduced the number of M2 macrophages. Compared with other groups, the expression levels of CD80 and CD86 on DCs in mice treated with FLA-OVA were significantly increased ( Fig.13 h, i). Accordingly, FLA-OVA expressed the highest level of SIINFEKL-H-2Kb peptide on the surface of DCs ( Fig.13 This is consistent with the results of in vitro experiments, indicating that FLA-OVA has a significant effect on the activation and maturation of antigen-presenting cells. + and CD8 + T cells are two major subsets of T cells, which play a crucial role in the defense against infection and cancer by producing different cytokines such as IFN-γ, IL-6, and TNF-α. Fig.13As shown in k and 1, FLA-OVA increased CD8 + In addition, the present invention found that interferon γ is the percentage of CD8 + T cells and CD4 + T cells stimulated by increased FLA-OVA ( Fig.13 FLA-OVA also triggered a strong antigen T cell response, as shown by the highest levels of CD8 + T cells ( Fig.13 In addition, Fig.13 As shown in the figure, Ki67 expression has an important effect on CD8 + T cells after treatment were compared with the FLA-OVA-boosted PBS group, indicating that FLA-OVA vaccination enhanced T cell proliferation. In summary, after subcutaneous immunization, FLA-OVA induced an effective cellular immune response in vivo. The above FLA-OVA-mediated increase in tumor retention and immune cell antigen uptake and the associated intratumoral immune activation, such as Figure 2 As shown in b.

[0078] Example 5

[0079] Inhibitory effect of FLA-OVA on tumor growth

[0080] The tumorigenicity and stability experiments were performed as follows: B16-OVA cells (1x 10 6 4 days after inoculation, the size of the tumor was measured with a caliper every 2 days. The length (L) and width (W) of each tumor were recorded, and the tumor volume (V) was calculated using the formula: V = L × W 2 / 2. Measured in cubic millimeters (mm 3 Tumor growth curves were drawn to evaluate tumor incidence, growth rate, and endpoint criteria. 3 At the end of the experiment, the mice were humanely euthanized and the tumor tissues were collected for photographic documentation.

[0081] The dose escalation test of FLA-OVA was performed as follows: B16-OVA cells (5x10 5 When the tumor reaches 100 mm 3 At about 24 hours, the mice were randomly divided into PBS, FLA-OVA (10 μg), FLA-OVA (30 μg), and FLA-OVA (100 μg) groups. The prepared FLA-OVA preparation with increasing doses was injected 4 times in a row at a dose of 50 μL / 2 days per tumor. PBS was used as the control group. The length (L) and width (W) of each tumor were recorded, and the tumor volume (V) was calculated using the formula: V = L × W2 / 2. Tumor tissues were collected and photographed after treatment. Tumor-draining lymph nodes, spleens, and tumors were dissected from vaccinated mice for immune response analysis.

[0082] OVA-specific IgG and IgG1 levels: 96-well flat-bottom polystyrene high-binding microplates were coated with OVA (100 μg / mL, 100 μL / well) in PBS at 4°C overnight. They were then washed three times with PBST buffer and blocked with 1% bovine serum albumin (BSA) in PBS and stored at room temperature for 1 hour. The serum was diluted 200-fold in PBS, added to the culture dish, and incubated at 37°C for 1 hour. They were washed three times with PBST buffer and incubated with enzyme-labeled goat anti-mouse IgG or IgG1 for 1 hour at room temperature. After washing with PBST, they were developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (200 μL / well) at room temperature. After 5 minutes of reaction, the reaction was stopped with stop solution (0.5 M H2SO4). Detection was performed at 450 nm using a microplate reader (BioTek Synergy neo2) for 30 minutes.

[0083] The experimental process of tumor treatment effect determination is as follows: B16-OVA cells (1×10 6 When the tumor reaches 70-80 mm 3 At about 14 days, the mice were randomly divided into PBS group, OVA group, FLA group and FLA-OVA group. Then FLA-OVA (FLA, 10 μg / mouse; OVA, 30 μg / mouse) was prepared, 50 μL per tumor, and injected intratumorally 4 times every 2 days. PBS, OVA, and FLA were used as control groups, and tumor-bearing mice were injected with equal amounts. The length (L) and width (W) of each tumor were recorded, and the tumor volume (V) was calculated using the following formula: V = L × W 2 / 2. After treatment, tumor tissues were collected and photographed. After fixation with 4% paraformaldehyde, immunofluorescence staining and H&E staining were performed to further evaluate the anti-tumor immune response and therapeutic effect of FLA-OVA.

[0084] After confirming the ability to trigger antigen-specific immune responses, the present invention then explored the potential of FLA-OVA to treat tumors. Since the dose-escalation response is one of the key parameters for evaluating the therapeutic effect, the immune response and tumor size under different doses of FLA-OVA were first evaluated. When the tumor volume reached about 100 mm 3B16-OVA tumor-bearing mice were intratumorally injected with 50 μL PBS, FLA-OVA (FLA, 10 μg; OVA, 10 μg), FLA-OVA (FLA, 10 μg; OVA, 30 μg), FLA (10 μg; OVA, 100 μg), 4 times every other day. Two days after the last vaccination, the levels of OVA-specific immunoglobulin G (IgG) and its subtype IgG1 in plasma were evaluated. IgG can bind to Fcγ receptors (Fcγrs) and subsequently induce antibody-dependent solid cell-mediated cytotoxicity. Most FDA-approved tumor therapeutic antibodies participate in anti-tumor activity through IgG1. As Fig.14 As shown in a and b, IgG1 levels increased with increasing amounts of OVA. There was no significant difference in serum IgG levels between the 30 μg and 100 μg FLA-OVA groups. In addition to antibody-mediated immunity, 30 μg FLA-OVA showed the most effective effect in triggering antigen-specific cellular responses. Compared with the other treatments, the 30 μg FLA-OVA group had a significantly higher level of DC and OVA-specific CD8 + T cells have the highest antigen presentation level ( Fig.14 The increase in spleen weight further confirmed the effective initiation of the immune response ( Fig.14 e). While effectively initiating immune responses, it was shown that 4 treatments with 30 μg of FLA-OVA could effectively inhibit tumor growth ( Fig.14 (f, g).

[0085] Then, the effect of FLA-OVA in inducing antitumor immune response in tumor-bearing mice was systematically tested. B16-OVA cells were subcutaneously injected into the right flank of C57BL / 6 mice to establish a mouse tumor-bearing model. The continued growth of the tumor demonstrated the tumorigenicity and stability of B16-OVA cells ( Fig.15 When the tumor volume reaches ~100mm 3 At the same time, 50 μL PBS, OVA (30 μg), FLA (10 μg) and FLA-OVA (containing equivalent FLA and OVA) were administered intratumorally to B16-OVA-bearing mice. Fig.17 In the plan of A, the tumor and tumor-draining lymph nodes were removed to evaluate the induced immune response. + and CD8 + The accumulation of T cells is essential for controlling tumor growth. Impressively, FLA-OVA treatment promoted the accumulation of CD4 + and CD8 + Tumor infiltration of lymphocytes including T cells ( Fig.16 (a) IFNγ in tumor samples from mice treated with FLA-OVA + CD4+ The number of T cells was 1.5-fold and 2-fold higher than that of mice treated with OVA and FLA, respectively. Fig.17 Consistent with the results obtained from tumor tissues, FLA-OVA also enhanced the expression of IFNγ in tumor-draining lymph nodes. + CD4 + T cell accumulation ( Fig.17 d, e). These stimulatory effects on IFNγ + CD8 + This is more evident in the activation of T cells. Fig.18 As shown in middle a, compared with the control group OVA and FLA, intratumoral IFNγ + CD8 + The number of T cells increased by about 2.5-fold. Similarly, FLA-OVA enhanced the expression of IFNγ in tumor-draining lymph nodes. + CD8 + T cell accumulation ( Fig.18 (b) It is noteworthy that OVA-specific CD8 + The frequency of T cells increased by 2-fold ( Fig.18 c), indicating its ability to enhance specific anti-tumor immune responses.

[0086] Next, we measured the value of FLA-OVA in treating tumors. Tumor cells were inoculated on day 7, and when the tumor size reached ∼80 mm on day 0, 3 PBS, OVA, FLA and FLA-OVA were injected in 50 μL PBS, i.e., FLA-OVA (FLA 10 μg, OVA 30 μg) was injected intratumorally every other day for a total of 4 times (eg Fig.19 PBS, OVA 30μg, and FLA 10μg were used as controls. Tumor size and mouse weight were recorded every other day after treatment. The mice were euthanized on the 10th day, and the tumor tissues were sliced ​​and photographed. Fig.16 As shown in middle bg, the tumor volume in the PBS group expanded uncontrollably. Although the tumor growth was slower than that in the PBS group, the tumor volume continued to increase after OVA treatment. Injection of FLA also had a significant inhibitory effect on tumor growth, which may be due to its innate immunogenicity that activates the immune response. Notably, among all treatments, FLA-OVA showed the most effective tumor growth inhibition, reflecting the complete elimination of 1 / 2 of the tumor. Hematoxylin and eosin (H&E) staining showed that FLA-OVA induced the greatest degree of tumor necrosis ( Fig.16 At the same time, FLA-OVA has a high tolerance to mice because the body weight of mice remained stable after treatment ( Fig. 20). H&E staining further confirmed the biosafety of FLA-OVA. After a period of FLA-OVA treatment, heart, liver, spleen, lung, and kidney tissue sections showed normal cell morphology ( Fig.21 ). The above results prove that FLA-OVA can effectively inhibit tumor growth without obvious toxic side effects.

[0087] Example 6

[0088] Preventive effect of FLA-OVA on tumor metastasis

[0089] The experimental process of preventing tumor metastasis was as follows: 6-week-old C57BL / 6 mice were immunized with different formulations such as PBS, OVA (30 μg / mouse), FLA (10 μg / mouse), and FLA-OVA (FLA, 10 μg / mouse); 1×10 6 B16-OVA cells (30 μg per mouse) were added. Lungs and spleens were harvested 14 days after inoculation. The number of lung metastatic nodules was recorded and the spleen was weighed. Lung sections were stained with H&E for metastasis analysis.

[0090] Encouraged by the inhibition of tumor growth, the efficacy of FLA-OVA in preventing tumor metastasis was further evaluated. C57BL / 6 mice were immunized with FLA-OVA or corresponding controls and then Fig. 22 The plan in a was to intravenously inject 1×106 B16-OVA cells. Lung tissue was collected 14 days after inoculation to detect tumor metastasis. Fig. 22 Middle b, Fig.23 As shown, the lung metastasis rate of mice treated with PBS and OVA was 100%. FLA slightly reduced the lung metastasis rate to 89%, which was further reduced to 67% after FLA-OVA treatment. Metastasis was then measured by examining the tumor nodules in the lungs. OVA treatment had little inhibition of metastasis compared to PBS, as both developed dense tumor nodules in the lungs. Similarly, the number of metastases decreased after FLA treatment, and FLA-OVA treatment could further reduce metastasis, with only sporadic lung metastatic nodules in treated mice ( Fig. 22 b, c). It was calculated that the number of metastatic nodules was reduced by 62 times compared with FLA treatment. This is consistent with the results of H&E staining. No obvious metastasis was observed in lung tissue after FLA-OVA immunization ( Fig. 22 In contrast, multiple tumor nodules were found in the other groups. In addition, the spleen weight of the mice in the FLA-OVA group increased significantly, which was due to the stimulation of a strong systemic immune response ( Fig. 22 These results suggest that FLA-OVA, in addition to its therapeutic effect on tumors, can also induce long-lasting specific anti-tumor immunity and prevent tumor metastasis.

[0091] Example 7

[0092] Necessity of adjuvant-antigen integration in FLA-OVA

[0093] To determine whether the adjuvant-antigen integration was redundant, we next systematically compared the antitumor effects of FLA-OVA with a simple mixture of FLA and OVA (FLA+OVA). Fig.24 As shown in a and b, at the end of treatment, the size of B16-OVA melanoma in the FLA-OVA group was reduced by about 3 times compared with the FLA+OVA group. H&E staining showed that FLA-OVA induced extensive tumor necrosis, while FLA+OVA could only induce small areas of necrosis ( Fig.24 (c) The significant effect of FLA-OVA in inhibiting tumor progression is related to the effective initiation of anti-tumor immune response. Fig.24 As shown in Figure d, FLA-OVA treatment enhanced the maximum infiltration of CD8+T cells in the tumor. In addition to inhibiting tumor growth, the present invention further evaluated the preventive effect of FLA+OVA and FLA-OVA on tumor metastasis. As expected, FLA-OVA treatment had the best effect in inhibiting tumor metastasis in all groups ( Fig.25 (a, b). The lung metastasis rate was 100% in the PBS group, 83% in the FLA+OVA group, and completely absent in the FLA-OVA group. Spleen enlargement, which contains highly activated T cells, was only observed in mice treated with FLA-OVA, indicating that FLA-OVA is more potent in activating immune responses than FLA+OVA ( Fig.25 c). The well-tolerated FLA-OVA inhibits melanoma growth and prevents melanoma lung metastasis. Figure 2 As shown in c.

[0094] Example 8

[0095] Versatility of FLA-based nanofiber vaccines

[0096] The versatility of vaccine development platforms is critical to addressing the difficulties of rapidly evolving mutants and the high cost and long cycle of vaccine development. Therefore, the present embodiments turn their attention to exploring the flexibility of flagella-based nanofiber vaccines to load other antigens. The S1 subunit of the spike protein that mediates SARS-CoV-2 recognition and entry into host cells was used as a model viral antigen. Similarly, a FLA-based nanofiber vaccine loaded with S1 protein (called FLA-S1) was prepared by replacing OVA with a viral antigen ( Fig.26 Detailed characterization data are shown in Figure 2a). Fig. 27As shown, it shows that the preparation of FLA-S1 was successful. The vaccine was injected subcutaneously on days 0 and 14, and spleen tissues of treated mice were collected on day 21 to measure the immune activation effect of FLA-S1 ( Fig.26 It is noteworthy that after FLA-S1 vaccination, the expression level of B220 antigen in the spleen was the highest, which means the accumulation of B cells ( Fig.26 (c) It is important to note that the formation of germinal centers (GC) in the spleen is essential for the long-term survival of B cell-dependent humoral immunity. + GC B cells expressed up-regulated, while other groups showed negative expression ( Fig.26 Meanwhile, after immunization with FLA-S1, the expression of CD69 on B cells was the highest ( Fig.26 e), indicating that B cell proliferation was promoted after immunization. Next, the present invention evaluated the activation of S1-specific B cells, which is essential for inducing an immune response mediated by S1-specific antibodies. Compared with the PBS and S1 groups, the FLA-S1 group showed a higher percentage of S1-specific B cells ( Fig.26 f). T cell immunity mediated by SARS-CoV-2S protein is more durable and robust than humoral immunity mediated mainly by B cells. The present invention further describes the effect of FLA-S1 on T cells. Fig.26 As shown in Figure g, the number of T cells in the FLA and FLA-S1 groups increased, indicating that T cells were activated. More importantly, FLA-S1 activated S1-specific T cells most significantly, 11 times higher than the S1 group ( Fig.26 (h) After vaccination with FLA-S1 vaccine, S1-specific CD4 + T cells and CD8 + T cell subsets also increased significantly ( Fig.26 (i, j). The viral antigen-specific immune responses elicited highlight the versatility of FLA as a nanofiber platform for developing different types of antigen-loaded vaccines.

[0097] Bacteria, as natural immunogenic compounds, especially rich pathogen-associated molecular patterns, can effectively activate immune cells and trigger systemic immune responses, and have been widely used in vaccine development. More attractively, bacteria-based vaccines show significant therapeutic potential in the treatment of cancer because they preferentially colonize and proliferate in hypoxic tumors. Bacterial vaccines carrying specific antigens are able to trigger specific anti-tumor immune responses. However, the clinical application of bacteria as therapeutic agents faces major challenges due to the thorny issues of infection-related side effects and disappointing overall treatment results. These concerns are related to the unclear and complex components of bacteria-based therapeutics, and alternative strategies need to be developed. In the above embodiments, the development of vaccines using bacterial FLA-based nanofibers is described. Unlike bacteria as a whole or various bacterial derivatives such as cell walls and extracellular vesicles, which are complex and unclear components, FLA is composed only of flagellin, which has been considered to be one of the important adjuvants for activating innate and adaptive immunity. FLA-based nanofiber vaccines are prepared by coating antigenic proteins on the surface of FLA. That is, the positively charged FLA nanofibers first self-assemble the cationic PEI on the surface through electrostatic interactions, and then further coat the antigen on the nanofibers with reversed surface charges to form layers of nanofiber structures. Note that in this system, FLA acts as both a delivery carrier and an immune adjuvant. The above examples demonstrate that the FLA-based nanofiber vaccine has strong immunogenicity in promoting cellular uptake of antigens, which further enhances antigen presentation by DCs and polarization of macrophages. Using OVA as a model tumor antigen, the significant ability of FLA-OVA to induce a strong tumor-specific immune response was shown in the B16-OVA tumor-bearing mouse model, effectively inhibiting tumor growth. In addition, in the B16-OVA mouse melanoma lung metastasis model, FLA-OVA showed significant tumor metastasis prevention ability. Importantly, the toxic damage to normal tissues caused by the administration of FLA-OVA was negligible, verifying the high tolerance for in vivo implementation. By replacing OVA with a viral antigen of the S1 protein, the flexibility of the FLA-based nanofiber platform in developing different types of antigen-loaded vaccines was further demonstrated. In view of these attractive advantages, the above-described embodiments of the present invention provide a unique paradigm with simplicity, high efficiency and good safety of vaccination.

[0098] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various modifications or variations within the scope of the claims, which does not affect the essence of the present invention. The above preferred features can be used in any combination without conflicting with each other.

Claims

1. A flagella nanofiber vaccine, characterized in that: It comprises flagella, cationic polymer and antigen, wherein the flagella are negatively charged, the cationic polymer is self-assembled on the surface of the negatively charged flagella through electrostatic action to form a nanofiber structure, and the antigen is coated on the surface of the nanofiber structure.

2. The flagella nanofiber vaccine according to claim 1, characterized in that The flagella serve as both a delivery vehicle and an immune adjuvant.

3. The flagella nanofiber vaccine according to claim 1, characterized in that The cationic polymer is PEI.

4. The flagella nanofiber vaccine according to claim 1, characterized in that The cationic polymer is chitosan or PMMA.

5. The flagella nanofiber vaccine according to claim 1, characterized in that The antigen is OVA.

6. The flagella nanofiber vaccine according to claim 1, characterized in that The antigen is a viral antigen of the S1 protein.

7. A method for preparing the flagella nanofiber vaccine according to any one of claims 1 to 6, characterized in that: include: providing a flagellum, the flagellum being negatively charged; Providing a cationic polymer, and self-assembling the cationic polymer on the surface of negatively charged flagella through electrostatic interaction to obtain a nanofiber structure with reversed surface charge; The flagellar nanofiber vaccine is obtained by coating antigens on the nanofiber structure with reversed surface charges.

8. Use of the flagella nanofiber vaccine according to any one of claims 1 to 6 in the preparation of a drug for preventing and / or treating a disease.

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