Nanometer fibrous adjuvant and application thereof

Abstract

The invention provides a nanofiber adjuvant and application thereof, and belongs to the technical field of biological medicines and vaccines. The nano fibrous adjuvant disclosed by the invention is formed by connecting muramyl dipeptide and flexible polypeptide, and the flexible polypeptide is glycine-glycine-phenylalanine-phenylalanine-naphthalene ring. The C end of the muramyl dipeptide molecule and the N end of the GGFF-Nap polypeptide are subjected to dehydration condensation to form a peptide bond, the bis-glycine is used as a flexible linker, and the bis-phenylalanine-naphthalene ring provides aromatic nucleus interaction. Under the assistance of GGFF-Nap, the muramyl dipeptide can be self-assembled into the nanofiber adjuvant with a nanofiber structure. The nanofiber adjuvant can precisely regulate and control the fate of B cells through multivalent co-display antigens, prepare long-acting antibody response vaccines, prolong the antibody response period and provide service for improving the durability of the vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and vaccine technology, specifically relating to a nanofiber adjuvant and its application. Background Technology

[0002] The persistence of prophylactic vaccines has been a focal point in global public health events. The persistence of vaccine-induced antibody responses is associated with long-lived plasma cells that continuously secrete antibodies. However, the insufficient number of long-lived plasma cells limits their role in maintaining a potent and durable antibody response. Post-vaccination, the recognition of antigens / adjuvants and their corresponding receptors may influence B cell fate differentiation towards long-lived plasma cells. The antigen / adjuvant display pattern is closely related to the strength of their receptor recognition. Multivalent display of antigens can promote antigen uptake by antigen-presenting cells, while multivalent display of adjuvants further provides co-stimulatory signals, thereby promoting B cell fate selection. In summary, multivalent co-display of antigens and adjuvants is likely to influence the production and maintenance of long-lived plasma cells, thereby enhancing antibody responses and achieving long-lasting protection.

[0003] There is an urgent need in this field to develop a vaccination strategy that increases the formation of long-lived plasma cells in the bone marrow, which would provide a potential solution for improving vaccine durability. Summary of the Invention

[0004] The purpose of this invention is to provide a nanofiber adjuvant and its application, which can precisely regulate B cell fate through multivalent co-display of antigens, ultimately expanding the long-lived plasma cell pool and prolonging the antibody response cycle.

[0005] This invention provides a nanofiber-like adjuvant, the chemical structural formula of which is: .

[0006] The present invention also provides the application of the nanofiber adjuvant in the preparation of long-acting antibody-response vaccine products.

[0007] Preferably, the vaccine comprises a novel coronavirus protein vaccine.

[0008] Preferably, the long-acting effect means that, compared with the first injection of the vaccine, a second injection of the vaccine at an interval of at least 336 days can produce higher antibody and neutralizing antibody titers.

[0009] Preferably, the vaccine comprises the novel coronavirus RBD protein and the nanofiber adjuvant.

[0010] Preferably, the mass ratio of the novel coronavirus RBD protein to the nanofiber adjuvant is 1:2~4.

[0011] The present invention also provides a novel coronavirus protein vaccine comprising the novel coronavirus RBD protein and the nanofiber adjuvant.

[0012] The present invention also provides the use of the nanofiber adjuvant or the novel coronavirus protein vaccine in the preparation of products that enhance the cellular immune response to the COVID-19 protein vaccine.

[0013] Preferably, the enhancement of the COVID-19 protein vaccine to the cellular immune response includes promoting the endosome membrane localization of NOD2.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a nanofiber-like adjuvant, chemically composed of a muramyl dipeptide (MDP) and a flexible polypeptide, glycine-glycine-phenylalanine-phenylalanine-naphthyl ring (GGFF-Nap). The C-terminus of the MDP molecule and the N-terminus of the GGFF-Nap polypeptide form a peptide bond through dehydration condensation, with diglycine acting as a flexible linker and the diphenylalanine-naphthyl ring providing aromatic ring interactions. With the assistance of GGFF-Nap, MDP can self-assemble into a nanofiber-like adjuvant, F-MG, possessing a nanofiber structure. This nanofiber-like adjuvant can promote the endosomal localization of NOD2, enabling the preparation of long-acting antibody-response vaccines using F-MG, thus prolonging the antibody response cycle and contributing to improved vaccine durability. Attached Figure Description

[0015] Figure 1 This is a chemical structure diagram of the nanofiber adjuvant F-MG in Example 1.

[0016] Figure 2 The image shows the time-of-flight mass spectrum of the nanofiber adjuvant F-MG in Example 1.

[0017] Figure 3 The circular dichroism chromatogram of the nanofiber adjuvant F-MG in Example 1.

[0018] Figure 4 This is a transmission electron microscope image of the self-assembled nanofiber adjuvant F-MG from Example 1.

[0019] Figure 5 The results are the surface plasmon resonance measurements between the F-MG adjuvant and the RBD protein in Example 1.

[0020] Figure 6 This is a schematic diagram of the chemical synthesis process of the nanofiber adjuvant F-MG in Example 1.

[0021] Figure 7This is a schematic diagram of the process of self-assembly of the nanofiber adjuvant F-MG in Example 1.

[0022] Figure 8 The results of F-MG's multivalent display of MDP promoting the interaction between MDP and NOD2 in Example 1 are shown. In Example 1, a represents the Western blot (WB) results of NOD2 expression in dendritic cells (DCs) stimulated by F-MG and MDP; b represents the average of the WB grayscale values; c represents the quantitative localization of NOD2 (green) on the endosomal membrane (red) using confocal microscopy and ImageJ, with cell nuclei stained with DAPI (blue). Statistical calculations were performed using one-way ANOVA and Dunnett's multiple comparison test. Error bars represent SEM images. "This represents P < 0.05;" "" represents P<0.01.

[0023] Figure 9 The results of the activation of NOD2-related downstream signaling pathways by the nanofiber vaccine prepared by the nanofiber adjuvant F-MG in Example 1 are shown. In the figure, a is a schematic diagram of the immunization procedure and sampling. Each mouse received two immunizations, two weeks apart, and spleen cells were taken for transcriptome sequencing. b is a co-expression network of differentially expressed genes. c is a schematic diagram of NOD2-related downstream signaling pathways.

[0024] Figure 10 This provides an additional result for the phagocytosis of DC antigens in Example 1.

[0025] Figure 11 The results of F-MG promoting responses to antibodies, neutralizing antibodies, memory B cells (MB), and long-lived plasma cells (LLPC) in Example 1 are shown below. a) is a schematic diagram of the immunization procedure and sampling, with each mouse receiving two immunizations two weeks apart; b) shows the titers of RBD-specific IgG and IgG subclasses (IgG1, IgG2a, IgG2b, IgG3) in serum samples measured by ELISA; c) shows the detection results of neutralizing antibody (nAb) levels against the original SARS-CoV-2 strain; d) shows the detection results of neutralizing antibody (nAb) levels against the SARS-CoV-2 XBB strain; e) shows a flow cytometry plot (left) and MB proportion (right); f) is a schematic diagram of RBD-specific bone marrow plasma cells (BMPC) ELIspots; g) shows a representative ELIspot well plot (left) and the number of RBD-specific BMPCs (right). Statistical significance was determined using one-way ANOVA and Dunnett's multiple comparison test. All data are expressed as mean ± SEM. "This represents P < 0.05;" "This represents P < 0.01;" "This means P < 0.001.

[0026] Figure 12 The nAb reaction results of F-MG with aluminum adjuvant in Example 1 are shown; statistical significance was analyzed using the t-test method. All data are expressed as mean ± SEM. "This represents P < 0.05;" "" represents P<0.01.

[0027] Figure 13 The results of F-MG+RBD enhanced T cell response in Example 1 are shown, where a is a schematic diagram of the immunization procedure and sampling; b is CD4. + Flow cytometry images (left) and statistical plots (right) of IL-2, TNF-α, and IL-4 expression in T cells; c represents CD8. + Flow cytometry images (left) and statistical plots (right) of IL-2, TNF-α, and L-4 expression on T cells; statistical significance was determined using one-way ANOVA and Dunnett's multiple comparison test. All data are expressed as mean ± SEM. "This represents P < 0.05;" "This represents P < 0.01;" "Represents P < 0.001", "This means P < 0.0001.

[0028] Figure 14 The results of F-MG+RBD-induced durable and recall immune responses in Example 1 are shown below. a) is a schematic diagram of the immunization procedure and sampling; each mouse received three immunizations at 2-week intervals, with a fourth immunization 336 days after the first injection. Blood samples were collected at 35, 49, 119, 329, and 347 days after the first injection. One week after the fourth immunization, spleen, lymph nodes (LN), and bone marrow (BM) were collected for immunoassay. b) shows the RBD-specific antibody response detected by ELISA. c) shows the ELIspot representative well diagram (left) and the number of RBD-specific BMPCs (right), respectively. d) shows the titers of RBD-specific IgG and its four IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3) in serum samples at 347 days, determined by ELISA. e) shows the results of the sham neutralization assay for the original SARS-CoV-2 strain neutralizing antibody (nAb) level. f) shows the results of the sham neutralization assay for the SARS-CoV-2 XBB strain neutralizing antibody (nAb) level. g) shows the CD4+ level. +Flow cytometry images (left) and statistical plot (right) of TNF-α and IFN-γ expression in T cells; h represents CD8. + Flow cytometry images (left) and statistical graphs (right) of TNF-α and IFN-γ expression on T cells; statistical significance was determined using one-way ANOVA and Dunnett's multiple comparison test. All data are expressed as mean ± SEM. "This represents P < 0.05;" "This represents P < 0.01;" "Represents P < 0.001", "This means P < 0.0001.

[0029] Figure 15 The results show the biocompatibility of F-MG+RBD in Example 1, where a is a schematic diagram of the immunization procedure and sampling; b is the mouse weight gain; c is the mouse body temperature; d is the mouse organ coefficient; e is the biochemical index of serum samples; and f is an HE staining image of pathological sections of the heart, liver, spleen, lung, and kidney, with a scale bar of 50 μm. Detailed Implementation

[0030] This invention provides a nanofiber-like adjuvant, which is composed of a muramyl dipeptide and a flexible polypeptide linked together, wherein the flexible polypeptide is glycine-glycine-phenylalanine-phenylalanine-naphthalene ring. The chemical structural formula of the nanofiber-like adjuvant is as follows: .

[0031] In this invention, the nanofiber-like adjuvant is abbreviated as F-MG. MG is chemically composed of a muramyl dipeptide (MDP) and a flexible polypeptide, glycine-glycine-phenylalanine-phenylalanine-naphthalene ring (GGFF-Nap). The C-terminus of the MDP molecule and the N-terminus of the GGFF-Nap polypeptide form a peptide bond through dehydration condensation, with diglycine acting as a flexible linker and the diphenylalanine-naphthalene ring providing aromatic ring interactions. With the assistance of GGFF-Nap, MDP can self-assemble into F-MG with a nanofiber structure. Gly-Gly-Phe-Phe-Napthylene (GGFF-Nap) can drive β-sheet self-assembly. Phenylalanine-phenylalanine (FF) is the functional unit that induces assembly into a β-sheet, the naphthalene ring (Nap) provides aromatic-aromatic interactions (II-I stacking) to promote assembly, and glycine (G) acts as a linker to regulate the flexibility of β-sheet arrangement. The chemical formula of MDP is C2. 19 H 32 N4O 11 The molecular weight is 492.48; the chemical formula of GGFF-Nap is C2. 33 H 35 N5O4 has a molecular weight of 565.67; the chemical formula of F-MG is C 52 H 65 N9O 14 The molecular weight is 1040.48. The nanofiber-like adjuvant described in this invention can bind to RBD to exert a delivery effect and promote NOD2 expression more effectively than MDP.

[0032] This invention also provides the application of the nanofiber adjuvant in the preparation of long-acting antibody-response vaccine products. In this invention, the vaccine is preferably a novel coronavirus protein vaccine. The vaccine preferably comprises novel coronavirus RBD protein and the nanofiber adjuvant. In the vaccine, the mass ratio of the novel coronavirus RBD protein to the nanofiber adjuvant is preferably 1:2 to 4, more preferably 1:2 to 3, and most preferably 1:2. In specific application, the MDP content in the nanofiber adjuvant is 10 μg. The long-acting nature of this invention means that, compared with the initial injection of the vaccine, a re-injection of the vaccine at an interval of at least 336 days can produce higher antibody and neutralizing antibody titers.

[0033] The present invention also provides a novel coronavirus protein vaccine comprising the novel coronavirus RBD protein and the nanofiber adjuvant.

[0034] The present invention also provides the use of the nanofiber adjuvant or the novel coronavirus protein vaccine in the preparation of products that enhance the cellular immune response of the COVID-19 protein vaccine. The enhanced cellular immune response of the COVID-19 protein vaccine includes promoting the endosome membrane localization of NOD2.

[0035] The nanofiber adjuvant described in this invention can be used to prepare long-acting antibody response vaccines, prolonging the antibody response period and providing services for improving vaccine durability.

[0036] The references involved in the following embodiments of the present invention are as follows: [1].Zhang,Y,Wang, R, He, C, et al. Amantadine-assemblednanostimulator enhances dimericRBDantigen-elicited cross-neutralizationagainst SARS-CoV-2 strains [J]. Nano Today, 2022, 43: 101393. [2].Li, W, Balachandran, YL, Hao, Y, et al. Amantadine surface-modified silver nanorods improves immunotherapy of HIV vaccine against HIV-infected cells [J]. ACS applied materials&interfaces, 2018, 10(34): 28494-28501. Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0038] Example 1 I. Experimental Materials and Methods 1. Design and construction of self-assembled nanofiber adjuvant F-MG 1.1 Synthesis and Characterization of F-MG Adjuvant The F-MG adjuvant was synthesized by Wuxi AppTec Co., Ltd. The final product was purified by high-performance liquid chromatography to obtain F-MG. A schematic diagram of the chemical synthesis process and self-assembly flow is shown below. Figure 6 , Figure 7 .

[0039] The morphology of the dissolved product was characterized using TEM (Tecnai G2 F20 U-TWIN TEM system, FEI, USA), and circular dichroism (CD) analysis was performed using a BioLogic system (MOS-450).

[0040] 1.2 Binding efficiency of F-MG and RBD 1.2.1 Experimental Materials For RBD expression and purification methods, please refer to the methods of Zhang, Y, et al. [1] The CM5 chip and related reagents required for surface plasmon resonance (SPR) included biacore maintenance kit type 2, aminecoupling kit, Acetate at pH 4.0, 4.5, 5.0, and 5.5, NaOH, and PBS-P, all purchased from Cytiva; DMSO was purchased from Sigma.

[0041] 1.2.2 Experimental Methods The interaction between MG or MDP and RBD was studied using a Biacore T200 (Washington, DC, USA).

[0042] Using 5% DMSO PBS-P as the run buffer, RBD was captured onto the CM5 chip as a ligand using an amine coupling kit. Analytes MG or MDP were diluted in the run buffer at concentrations ranging from 0.3125–10 μM and 3.123–200 μM, respectively, and the solutions were set to flow through the chip channels containing the captured RBD. Response units (RUs) were recorded. The sensor chip surface was then regenerated using 50% DMSO at a time of 150 s, with binding time set at 120 s and dissociation time at 180 s. Dose-response data were collected using a conventional multi-cycle method, and the data were automatically fitted to a 1:1 binding pattern for kinetic and steady-state affinity calculations.

[0043] 2. The multivalent display of MDP by F-MG facilitates the interaction between MDP and NOD2. 2.1 WB Analysis 2.1.1 Experimental Materials 5× protein loading buffer and TBS were purchased from Servicebio, markers from Vazyme, and reagents required for gel preparation, including 30% Acrylamide / Bis solution (29:1), 1M Tris-HCl (pH 8.8 / 6.8), 10% SDS, ammonium persulfate (AP), and TEMED, were purchased from Solarbio. Electrophoresis buffer and transfer buffer were purchased from Biosharp, PVDF membranes from Millipore, anti-mouse NOD2 antibody and ECL from Affinity, internal control anti-mouse HSP60 antibody from CST, horseradish peroxidase (HRP) labeled secondary antibody from Servicebio, and RPMI 1640 medium and fetal bovine serum (FBS) from Gibco.

[0044] 2.1.2 Experimental Methods DC2.4 cells were cultured in RPMI 1640 + 10% FBS in a 5% CO2 incubator at 37°C. They were divided into three groups: Blank (medium), F-MG (2 μg, containing 1 μg MDP), and MDP (1 μg). After 6 hours of incubation, the supernatant was removed, and the cells were washed three times with PBS. Cells were scraped directly with 1× protein loading buffer, boiled to denature them, and then subjected to electrophoresis, membrane transfer, incubation with antibodies (Anti-mouse NOD2 was used at a 1:2000 dilution, and anti-mouse HSP60 was used at a 1:1000 dilution) and color development to complete Western blotting.

[0045] 2.2 Fluorescence confocal microscopy imaging 2.2.1 Experimental Materials Anti-mouse NOD2 antibody was purchased from Affinity. Red fluorescently labeled mouse endosomal membrane protein antibody CoraLite555 anti-mouse CD107a / LAMP1 and green fluorescently labeled anti-mouse IgG antibody CoraLite488-conjugated goat anti-mouse IgG (H+L) were purchased from Proteintech. DAPI mounting medium was purchased from Invitrogen.

[0046] 2.1.2 Experimental Methods DC2.4 cell smears of appropriate density were divided into Blank (culture medium) and F-MG (2 μg, containing MDPI) culture medium. Three groups of cells were incubated for 4 hours: one group of CoraLite488-conjugated goat anti-mouse IgG (H+L) (1:200 dilution) and one group of CoraLite555 anti-mouse CD107a / LAMP1 (1:100 dilution). After incubation, the cells were incubated for 4 hours, the supernatant was removed, and the cells were washed twice with PBS pre-cooled to 4°C. Then, the cells were blocked at 37°C for 30 minutes with 5% FBS / PBS. The cells were then incubated overnight at 4°C with Anti-mouse NOD2 (1:200 dilution) and washed five times with ice-cold PBS. CoraLite488-conjugated goat anti-mouse IgG (H+L) (1:200 dilution) and CoraLite555 anti-mouse CD107a / LAMP1 (1:100 dilution) were mixed and incubated at 37°C for 1 hour. The cells were then washed three times with PBS at room temperature and placed on a glass slide. Finally, DAPI mounting medium was added, and the cells were observed and photographed using a fluorescence confocal microscope (Leica SP8).

[0047] 3. F-MG effectively enhances the humoral immune response to the COVID-19 protein vaccine. 3.1 Animal Immunization 3.1.1 Laboratory animals SPF-grade 6-8 week old BALB / c mice were all purchased and bred at the Laboratory Animal Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences. All animal experiments were approved by the Animal Ethics Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences (DWSP202108 010). 3.1.2 Experimental Materials Aluminum adjuvant, provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0048] 3.1.3 Experimental Methods BALB / c mice aged 6-8 weeks were vaccinated intramuscularly twice, with a 2-week interval. All vaccines were prepared in physiological saline and divided into four groups: F-MG (20 μg, containing 10 μg MDP) + RBD (10 μg), MDP (10 μg) + RBD (10 μg), and RBD (10 μg). In the live virus neutralization experiment, mice were injected three times, with a 2-week interval, into two groups: F-MG (40 μg) + RBD (10 μg) and aluminum adjuvant (35 μg) + RBD (10 μg).

[0049] 3.2 IgG and its subtypes ELISA 3.2.1 Experimental Materials Coating buffer and tetramethyl benzidine (TMB) two-component chromogenic solution were purchased from Sorlabio. HRP-labeled anti-mouse IgG, PBS and PBST were purchased from Bioharp. Bovine serum albumin (BSA) was purchased from BioFroxx. Polystyrene ELISA plates (plates, 96 wells) were purchased from NEST. HRP-labeled anti-mouse IgG1, IgG2a, IgG2b and IgG3 were all purchased from Invitrogen.

[0050] 3.2.2 Experimental Methods Dilute RBD to a concentration of 5 μg / mL with coating buffer and add 100 μL to each polystyrene reaction well. Incubate overnight at 4°C, discard the solution, and add 250 μL / well of blocking buffer (2% PBS / BSA). Incubate at 37°C for 2 h, then discard the blocking buffer and wash three times with PBST. Add serum samples to each well and incubate at 37°C for 1 h. Wash three times with PBST. Add the corresponding HRP-labeled antibodies (anti-mouse IgG at a dilution of 1:40000, anti-mouse IgG1 at 1:20000, anti-mouse IgG2a and IgG2b at 1:2000, and IgG3 at 1:1000) at 100 μL / well. Incubate at 37°C for 1 h, then wash three times. Prepare TMB chromogenic solution fresh and add 100 μL / well to TMB chromogenic solution. Incubate at 37°C in the dark for 10 min. Add 100 μL of stop solution (2M sulfuric acid solution) to each well, and then read the values ​​on a microplate reader. Measure the OD value at 450 nm and 630 nm, respectively; the difference is the OD value of that well. Serum antibody titer is defined as the maximum dilution greater than 2.1 times the OD value of the negative control well (blank mouse serum).

[0051] 3.3 Pseudovirus Neutralization Experiment 3.3.1 Experimental Materials SARS-CoV-2 pseudovirus was purchased from Sino Biological, DMEM complete culture medium was purchased from Gibco, cell lysis buffer and luciferase assay reagent were both purchased from Promega, and 96-well black cell culture plates were purchased from Corning.

[0052] 3.3.2 Experimental Methods 50 μL of pseudovirus and serially diluted mouse serum were incubated in a Thermo Scientific cell culture incubator at 37°C with 5% CO2 for 1 h, and then 5 × 10⁻⁶ ACE2-overexpressing 293T cells were added. 4Cells were cultured in wells (samples / well) for 24 hours with DMEM. After lysing the cells with cell lysis buffer, luciferase substrate was added. The autoluminescence value was read in a multi-functional microplate reader (Bio-Tech) to determine the relative activity of luciferase. The inhibition rate was calculated using the formula [1 - (sample wells - blank control) / (virus control - blank control)] × 100%.

[0053] 3.4 Flow cytometry 3.4.1 Experimental Materials RPMI 1640 medium was purchased from Gibco; erythrocyte lysis buffer was purchased from Solarbio; 400-mesh nylon mesh was purchased from Taobao; the flow cytometry antibodies used and their sources are summarized in Table 1; and the live / dead dye (Zombie NIR™) was purchased from Biolegend.

[0054] Table 1 shows the antibodies used.

[0055] 3.4.2 Experimental Methods The obtained spleens were wrapped in 400-mesh nylon cloth and ground in a culture dish containing 5 mL of RMPI 1640 complete medium. The cell suspension was transferred to 15 mL centrifuge tubes and centrifuged (1800 rpm, 5 min, 4℃). The supernatant was discarded, and erythrocyte lysis was performed (2 mL of erythrocyte lysis buffer was added, the mixture was gently vortexed and lysed for no more than 5 min, then 5 mL of medium was added to stop the lysis and the mixture was stirred). The cells were centrifuged (1800 rpm, 5 min, 4℃), the supernatant was discarded, and the cells were resuspended in 3 mL of complete medium. All cell suspensions were counted and quantified to a value of 10. 6 Staining was then performed using Zombie NIR™ live / dead stain, incubated at room temperature in the dark for 15 min, followed by washing once with 2% FBS / PBS. Cell surface labeling was performed using the corresponding flow cytometry antibodies (as shown in Table 5), incubated at 4°C in the dark for 30 min, washed once with 2% FBS / PBS, and resuspended in 2% FBS / PBS. At least 100,000–200,000 lymphocytes were collected using a flow cytometer (BD LSR Fortessa, BD Biosciences), and the results were analyzed using FlowJo software (Tree Star, Ashland, OR).

[0056] 3.5 ELISpot of bone marrow plasma cells 3.5.1 Experimental Materials 96-well plates (PVDF membranes) were purchased from Millipore, biotin-labeled anti-mouse IgG was purchased from Bio-Sens, streptomycin-labeled AP was purchased from Invitrogen, DPBS was purchased from Gibco, and the BCIP / NBT colorimetric kit was purchased from Solarbio.

[0057] 3.5.2 Experimental Methods Pre-wet ELISpot plates with 15 μL of 35% ethanol per well for 2 min at room temperature, wash twice with PBS, add 100 μL of 5 μg / mL RBD to each well, incubate overnight at 4°C, wash twice with PBS, block with 10% FBS / RPMI 1640 at room temperature for 2 h, and then transfer the lysed bone marrow cell suspension (2 × 10⁻⁶ cells / well) to the plate. 6 Add 1 sample per well and incubate overnight at 4°C. Wash 3 times with PBST. Add 75 μL of 0.5 μg / mL biotinylated anti-mouse IgG antibody and incubate at room temperature for 2 h. Wash 6 times with PBST. Add streptomycin-labeled AP (1:500 dilution) and incubate at room temperature for 2 h. Wash 5 times with PBST and 1 time with DPBS. Add 100 μL of BCIP / NBT to each well for 7 min of color development. Stop the reaction with water. After the plate dries, count the spots using an ELISA dot imager (CTL).

[0058] 3.6 Live virus neutralization experiment 3.6.1 Experimental Materials The live SARS-CoV-2 virus and Vero-E6 cells were provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences, and the experiments were conducted in the biosafety level-3 (BLS-3) laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0059] 3.6.2 Experimental Methods Serum diluted twofold sequentially was mixed with live virus and incubated at 37°C for 1 hour. The mixture was then added to Vero-E6 monolayer cells, with each sample prepared in triplicate. Cytopathic effect (CPE) was observed daily in each well and recorded one week post-infection. The neutralizing titer of mouse antiserum for complete CPE prevention was calculated.

[0060] 4. F-MG effectively enhances the cellular immune response to the COVID-19 protein vaccine. 4.1 Animal Immunization 4.1.1 Laboratory Animals SPF-grade 6-8 week old BALB / c mice were all purchased and cultured at the Laboratory Animal Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0061] 4.1.2 Experimental Methods BALB / c mice aged 6-8 weeks were injected intramuscularly with the vaccine twice, with an interval of 2 weeks. All vaccines were prepared in physiological saline and divided into F-MG (20 μg, containing MDP 10 μg) + RBD (10 μg), MDP (10 μg) + RBD (10 μg) and RBD (10 μg) groups.

[0062] 4.2 Flow cytometry 4.2.1 Experimental Materials The flow cytometry antibodies used and their sources are summarized in Table 2; the fixation / perm solution, perm / wash solution, and flow cytometry sheath solution were all purchased from BD; the protein transport inhibitor Golgistop was purchased from BD, and Brefeldin A was purchased from Sigma; the flow cytometry positive stimulant (cell activation cocktail) and the live / dead dye (Zombie NIR™) were purchased from Biolegend.

[0063] Table 2 uses antibodies

[0064] 4.2.2 Experimental Methods Lymph nodes were wrapped in 400-mesh nylon cloth and ground in a culture dish containing 5 mL of RMPI1640 complete medium. The cell suspension was transferred to 15 mL centrifuge tubes, centrifuged (1800 rpm, 5 min, 4℃), the supernatant was discarded, 2 mL of PBS was added for resuspension, and the cells were centrifuged again (1800 rpm, 5 min, 4℃). The supernatant was discarded, and 3 mL of complete medium was added for resuspension. All cell suspensions were counted and quantified to 10-10. Cells were stimulated with 2 μg / mL RBD protein. DMSO was used as a negative control, and a positive stimulant was used as a positive control. Cells were incubated at 37℃ and 5% CO2 for 6 h in the presence of protein transport inhibitors. Staining was performed by incubation with ZombieNIR™ live / dead stain at room temperature in the dark for 15 min, followed by washing once with 2% FBS / PBS. Cell surface labeling was performed using the corresponding flow cytometry antibodies (as shown in Table 2), incubated at 4℃ in the dark for 30 min, followed by washing once with 2% FBS / PBS. Add fixation / permeabilization buffer and incubate at 4°C in the dark for 30 min, followed by one Perm / Wash wash. Perform intracellular staining with the appropriate intracellular factor flow cytometry antibody, incubate at 4°C in the dark for 30 min, and wash once with Perm / Wash. Resuspend in 2% FBS / PBS and collect at least 100,000-200,000 lymphocytes using a flow cytometer (BD LSR Fortessa, BD Biosciences). Analyze the results using FlowJo software (Tree Star, Ashland, OR).

[0065] 5. F-MG long-acting enhances the immune response to COVID-19 protein vaccines. 5.1 Animal Immunization 5.1.1 Laboratory Animals SPF-grade 6-8 week old BALB / c mice were all purchased and cultured at the Laboratory Animal Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0066] 5.1.2 Experimental Methods BALB / c mice aged 6-8 weeks were vaccinated intramuscularly with three initial injections, spaced two weeks apart. After 48 weeks of observation (after the first injection), a booster injection was administered intramuscularly. All vaccines were prepared in physiological saline and divided into four groups: F-MG (40 μg, containing 20 μg MDP) + RBD (10 μg), MDP (20 μg) + RBD (10 μg), and RBD (10 μg).

[0067] 5.2 IgG and its subtypes ELISA 5.2.1 Experimental Materials Coating buffer and TMB (tetramethyl benzidine) two-component chromogenic solution were purchased from Sorlabio. HRP-labeled anti-mouse IgG, PBS and PBST were purchased from Bioharp. Bovine serum albumin (BSA) was purchased from BioFroxx. Polystyrene ELISA plates (plates, 96 wells) were purchased from NEST. HRP-labeled anti-mouse IgG1, IgG2a, IgG2b and IgG3 were all purchased from Invitrogen.

[0068] 5.2.2 Experimental Methods Dilute RBD to a concentration of 5 μg / mL with coating buffer and add 100 μL to each polystyrene reaction well. Incubate overnight at 4°C, discard the solution, and add 250 μL / well of blocking buffer (2% PBS / BSA). Incubate at 37°C for 2 h, then discard the blocking buffer and wash three times with PBST. Add serum samples to each well and incubate at 37°C for 1 h. Wash three times with PBST. Add the corresponding HRP-labeled antibody (anti-mouse IgG at a dilution of 1:40000, anti-mouse IgG1 at 1:20000, anti-mouse IgG2a and IgG2b at 1:2000, and IgG3 at 1:1000) at 100 μL / well. Incubate at 37°C for 1 h, then wash three times. Prepare TMB chromogenic solution fresh and add 100 μL / well to TMB chromogenic solution. Incubate at 37°C in the dark for 10 min. Add 100 μL of stop solution (2M sulfuric acid solution) to each well, and then read the values ​​on a microplate reader. Measure the OD value at 450 nm and 630 nm, respectively; the difference is the OD value of that well. Serum antibody titer is defined as the maximum dilution greater than 2.1 times the OD value of the negative control well (blank mouse serum).

[0069] 5.3 Bone marrow plasma cells ELISpot 5.3.1 Experimental Materials 96-well plates (PVDF membrane) were purchased from Millipore, biotin-labeled anti-mouse IgG from Bio-Sens, streptomycin-labeled AP from Invitrogen, DPBS from Gibco, and the BCIP / NBT colorimetric kit from Solarbio.

[0070] 5.3.2 Experimental Methods Pre-wet ELISPOT plates with 15 μL of 35% ethanol per well for 2 min at room temperature, wash twice with PBS, add 100 μL of 5 μg / mL RBD to each well, incubate overnight at 4°C, wash twice with PBS, block with 10% FBS / RPMI 1640 at room temperature for 2 h, and then transfer the lysed bone marrow cell suspension (2 × 10⁻⁶ cells / well) to the plate. 6 Add 1 sample per well and incubate overnight at 4°C. Wash 3 times with PBST. Add 75 μL of 0.5 μg / mL biotinylated anti-mouse IgG antibody and incubate at room temperature for 2 h. Wash 6 times with PBST. Add streptomycin-labeled AP (1:500 dilution) and incubate at room temperature for 2 h. Wash 5 times with PBST and 1 time with DPBS. Add 100 μL of BCIP / NBT to each well for 7 min of color development. Stop the reaction with water. After the plate dries, count the spots using an ELISA dot imager (CTL).

[0071] 5.4 Pseudovirus Neutralization Experiment 5.4.1 Experimental Materials SARS-CoV-2 pseudovirus was purchased from Sino Biological, DMEM complete culture medium was purchased from Gibco, cell lysis buffer and luciferase assay reagent were both purchased from Promega, and 96-well black cell culture plates were purchased from Corning.

[0072] 5.4.2 Experimental Methods 50 μL of pseudovirus and serially diluted mouse serum were incubated in a Thermo Scientific cell culture incubator at 37°C with 5% CO2 for 1 h, and then 5 × 10⁻⁶ ACE2-overexpressing 293T cells were added. 4 Cells were cultured in wells (samples / well) for 24 hours with DMEM. After lysing the cells with cell lysis buffer, luciferase substrate was added. The autoluminescence value was read in a multi-functional microplate reader (Bio-Tech) to determine the relative activity of luciferase. The inhibition rate was calculated using the formula [1 - (sample wells - blank control) / (virus control - blank control)] × 100%.

[0073] 5.5 Flow Cytometry 5.5.1 Experimental Materials The flow cytometry antibodies used and their sources are summarized in Table 2; the fixation / perm solution, perm / wash solution, and flow cytometry sheath solution were all purchased from BD; the protein transport inhibitor Golgistop was purchased from BD, and Brefeldin A was purchased from Sigma; the flow cytometry positive stimulant (cell activation cocktail) and the live / dead dye (Zombie NIR™) were purchased from Biolegend.

[0074] 5.5.2 Experimental Methods Lymph nodes were wrapped in 400-mesh nylon cloth and ground in a culture dish containing 5 mL of 1640 complete medium. The cell suspension was transferred to 15 mL centrifuge tubes, centrifuged (1800 rpm, 5 min, 4°C), the supernatant was discarded, 2 mL of PBS was added for resuspension, and the cells were centrifuged again (1800 rpm, 5 min, 4°C), the supernatant was discarded, and 3 mL of complete medium was added for resuspension. All cell suspensions were counted and quantified to 10-10. Cells were stimulated with 2 μg / mL LRBD protein. DMSO was used as a negative control, and a positive stimulant was used as a positive control. Cells were incubated at 37°C and 5% CO2 for 6 h in the presence of protein transport inhibitors. Staining was performed by incubation with Zombie NIR™ live / dead stain at room temperature in the dark for 15 min, followed by washing once with 2% FBS / PBS. Cell surface labeling was performed using the corresponding flow cytometry antibodies (as shown in Table 2), incubated at 4°C in the dark for 30 min, followed by washing once with 2% FBS / PBS. Add fixation / permeabilization buffer and incubate at 4°C in the dark for 30 min, followed by one Perm / Wash wash. Perform intracellular staining with the appropriate intracellular factor flow cytometry antibody, incubate at 4°C in the dark for 30 min, and wash once with Perm / Wash. Resuspend in 2% FBS / PBS and collect at least 100,000-200,000 lymphocytes using a flow cytometer (BDLSR Fortessa, BDBiosciences). Analyze the results using FlowJo software (TreeStar, Ashland, OR).

[0075] 6. Biocompatibility of F-MG in COVID-19 protein vaccines 6.1 Animal immunization, monitoring of animal body weight, body temperature, tissue and organ coefficients, and serum biochemical indicators. 6.1.1 Laboratory Animals SPF-grade 6-8 week old BALB / c mice were all purchased and cultured at the Laboratory Animal Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0076] 6.1.2 Experimental Methods BALB / c mice aged 6-8 weeks were vaccinated once via tail vein. The vaccine was prepared in physiological saline and divided into two groups: F-MG (80 μg) + RBD (10 μg) and physiological saline. The mice were observed for 7 days, with daily monitoring of body weight changes. Body temperature monitoring began 1 hour before vaccination and continued at 0, 1, 2, 3, 4, 5 hours, and 1, 2, 3, 4, 5, 6, and 7 days. Serum samples were collected 7 days post-injection, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels were measured using a fully automated biochemical analyzer (BS-200, Mindray). The organs (heart, liver, spleen, lungs, kidneys, and brain) were weighed and recorded 7 days post-injection, and the organ coefficient (%) was calculated using the formula: Organ weight (%) = (Organ weight / Mouse body weight) x 100%.

[0077] 6.2 Immunohistochemical analysis 6.2.1 Experimental Materials The 4% paraformaldehyde solution was purchased from Biosharp.

[0078] 6.2.2 Experimental Methods Seven days after injection, organs (heart, liver, spleen, lung, and kidney) were collected, fixed in 4% paraformaldehyde, embedded in paraffin, cut to a thickness of 5 μm, stained with hematoxylin and eosin (HE), and observed under an optical fiber microscope.

[0079] 7. Statistical Analysis Methods 7.1.1 Software Use GraphPad Prism9 for data analysis and graphing.

[0080] 7.1.2 Statistical Methods For comparisons of three or more groups, one-way ANOVA was used, followed by Dunnett's multiple comparison test. For comparisons between two groups, the two independent samples t-test was used for statistical analysis, with P < 0.05 indicating statistical significance.

[0081] II. Experimental Results: 1. Design and construction of self-assembled nanofiber adjuvant F-MG In this invention, the carboxyl terminus of MDP and the amino terminus of GGFF-Nap are synthesized via an amide condensation reaction to form MDP-GGFF-Nap (MG). Utilizing the self-assembly driving force of GGFF-Nap, MG self-assembles into nanofibers (F-MG) in aqueous solution, exhibiting multivalent MDP and capable of binding to RBD proteins for delivery. The chemical structure of the synthesized F-MG adjuvant is as follows: Figure 1 As shown in the figure. Time-of-flight mass spectrometry results indicate that the purity of the chemically synthesized F-MG exceeds 98% ( Figure 2 ).

[0082] Transmission electron microscopy (TEM) results show the nanofiber structure of F-MG. Figure 4 The CD spectrum showed a positive peak at 197 nm (+ / - 10 nm) and a negative peak at 212 nm (+ / - 10 nm), which helped confirm the β-sheet conformation. Figure 3 We used surface plasmon resonance to determine the binding of RBD antigen to F-MG. The Kp value was 21.38 μM, significantly lower than that of MDP (0.01366 M), indicating that F-MG can bind to RBD and self-assemble into the immunoactivator MDP, thus enhancing its delivery function. The equilibrium dissociation constant (KD) between F-MG and RBD was 1.021 × 10⁻⁶. -6 M indicates that F-MG adjuvant and RBD protein can bind more efficiently and tightly. Figure 5 ).

[0083] 2. The multivalent display of MDP by F-MG facilitates the interaction between MDP and NOD2. Multivalent interactions involve multiple copies of a ligand on one surface simultaneously binding to multiple receptors on another surface. Multivalent interactions can generate higher affinity than monovalent interactions, an enhancement stemming from an increased "effective concentration"—the binding of one ligand to a receptor promotes the binding of other ligands to receptors. Benefiting from the peptide self-assembly platform, MDPs in F-MG nanofibers are multivalently displayed on the assembly surface, exhibiting higher affinity for the receptor NOD2. Cellular experiments verified that F-MG promotes NOD2 expression more effectively than MDP. Western blotting analysis of NOD2 expression in DC cells stimulated with F-MG or MDP showed that F-MG significantly promoted NOD2 expression. Figure 8 a, Figure 8 b). The endosome membrane localization of NOD2 is of great significance for the recognition of MDPs and the activation of downstream signaling pathways.

[0084] according to Figure 9 Nanofiber vaccines can effectively activate NOD2-related downstream signaling pathways, such as the MAPK pathway, NF-κB pathway, and TNF pathway, achieving good innate immune activation, indicating that F-MG has good adjuvant properties for immune stimulation.

[0085] Therefore, fluorescence confocal microscopy was used to observe the endosome membrane localization of NOD2 in DC cells stimulated by F-MG and MDP. The results showed that F-MG promoted the endosome membrane localization of NOD2 more effectively than MDP. Figure 8 c).

[0086] Figure 10The results showed that F-MG could increase antigen phagocytosis compared to MDP. These results demonstrate that F-MG can better activate NOD2 and exert immune activation function than MDP, laying a theoretical foundation for using F-MG as an adjuvant in COVID-19 protein vaccines.

[0087] 3. F-MG effectively enhances the humoral immune response to the COVID-19 protein vaccine. After discovering that F-MG can both bind to RBD to exert a delivery function and multivalently exhibit the immune activation effect of MDP, we evaluated F-MG as an adjuvant in COVID-19 protein vaccines. First, we evaluated the effect of F-MG on regulating RBD-induced humoral immune responses.

[0088] BALB / c mice aged 6-8 weeks were immunized twice by intramuscular injection at two-week intervals using F-MG+RBD, MDP+RBD, and RBD. One week after the full immunization, mouse serum, lymph node, and bone marrow single-cell suspensions were collected for evaluation. Figure 11 a). From the titers of IgG and IgG subtypes ( Figure 11 (b) The trends were consistent across the F-MG+RBD, MDP+RBD, and RBD groups, with F-MG+RBD inducing the highest antibody titers. Compared to RBD, F-MG+RBD significantly increased the titers of IgG, IgG1 (associated with Th2 responses), and IgG2a (associated with Th1 responses) (P<0.05), showing a more balanced Th1 / Th2 response compared to MDP+RBD, which tended to induce higher levels of IgG1 (associated with Th2 responses). Compared to MDP+RBD, F-MG+RBD elicited statistically significantly higher levels of IgG2a. IgG2a has been shown to be the main antibody type mediating antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in mice, playing a crucial role in viral clearance, indicating that F-MG can promote a robust antibody response to the vaccine. The pseudovirus neutralization experiment showed that F-MG+RBD induced the highest neutralizing antibody titer, and the neutralizing antibody level against the original SARS-CoV-2 strain induced by F-MG+RBD was significantly higher than that in the RBD group (P<0.01). Figure 11 c) It also had the highest mean cross-neutralizing antibody titer against the currently prevalent XBB strain of SARS-CoV-2. Figure 11 d). Memory B cells play a crucial role in durable cross-protection against emerging SARS-CoV-2 variants; flow cytometry results showed that F-MG+RBD induced the highest levels of memory B cell (MB) responses. Figure 11e); while long-lived plasma cells (LLPCs) are mainly found in the bone marrow, and the differences in the number of LLPCs can be analyzed by ELIspot results of antigen-specific bone marrow plasma cells. Figure 11 f), the results showed that the F-MG+RBD group also exhibited the highest number of bone marrow plasma cells (f). Figure 11 (g) Furthermore, the differences were statistically significant, and the high levels of response in memory B cells and bone marrow plasma cells together suggest that F-MG has the potential to provide sustained protection. In summary, F-MG demonstrates an adjuvant effect that can effectively enhance humoral immunity to COVID-19 protein vaccines.

[0089] To further clarify its effect level, F-MG was compared with the most widely used aluminum adjuvant. Both F-MG+RBD and Al+RBD were administered intramuscularly twice, two weeks apart, to 6-8 week old BALB / c mice. One week after full immunization, mouse serum was collected for live virus cross-neutralization experiments. The results showed that F-MG+RBD had significantly higher neutralizing antibody titers against the original SARS-CoV-2 strain, Delta, and Omicron strains. Figure 12 This further illustrates that F-MG can promote cross-protection in vaccines.

[0090] 4. F-MG effectively enhances the cellular immune response to the COVID-19 protein vaccine. F-MG+RBD, MDP+RBD, and RBD were administered intramuscularly to 6-8 week old BALB / c mice twice, with a two-week interval. One week after the full immunization, single-cell suspensions of the mouse spleen were collected for cellular immunity evaluation. Figure 13 a).

[0091] Compared with the RBD group, F-MG promoted CD4. + The expression of IL-2, TNF-α (related to Th1 response), and IL-4 (related to Th2 response) in T lymphocytes was statistically significant, with TNF-α and IL-4 showing statistically significant differences. Figure 13 b) F-MG exhibited a balanced Th1 / Th2 response. Compared with the MDP+RBD and RBD groups, the F-MG+RBD group showed a higher CD4 count. + T lymphocytes and CD8 + T lymphocytes all expressed more TNF-α, and the difference was statistically significant. Figure 13 c).

[0092] 5. F-MG long-acting enhances the immune response to COVID-19 protein vaccines. Based on the high levels of MB and LLPC responses in humoral immunity within a short period and the indication of sustained protection from high TNF-α expression in cellular immunity evaluation, we further observed the immune persistence and recall response of F-MG+RBD. BALB / c mice aged 6-8 weeks were injected intramuscularly three times at 2-week intervals with F-MG+RBD, MDP+RBD, and RBD. After 336 days (one injection), a booster injection was given. Seven days later, serum, lymph node, spleen, and bone marrow single-cell suspensions were collected to observe the immune recall response. Figure 14 a). At the observed time points, the highest RBD-specific IgG antibody titer was observed 21 days after the three-dose immunization, followed by a gradual decline. The antibody titer in the F-MG+RBD group declined more slowly. Seven days before the booster injection at day 336, the average antibody titer in the F-MG+RBD group was higher than that in the MDP+RBD and RBD groups at day 119. Seven days after the booster injection, the antibody titer in the F-MG+RBD group rapidly recovered and was higher than that in other groups. Figure 14 b). Long-lived plasma cells provide rapid and effective infection protection through pre-secreting antibodies, while memory B cells differentiate into plasma cells in response to reinvading antigens as a reserve. In short-term observation, the F-MG+RBD group exhibited a high level of memory B cell response. Figure 11 e), consistent with theory, the F-MG+RBD group also had the highest number of antigen-specific bone marrow plasma cells in the immune recall response 336 days later. Figure 14 c). We further analyzed the titers of IgG and IgG subtypes ( Figure 14 d) Similar to the short-term observation results, the trends of the F-MG+RBD, MDP+RBD, and RBD groups were consistent, with F-MG+RBD still inducing the highest antibody titer. Compared with RBD, F-MG+RBD significantly increased the titers of IgG1, IgG2a, and IgG2b, with statistically significant differences. Compared with MDP+RBD, F-MG+RBD stimulated a statistically significantly higher level of IgG2b, further demonstrating that F-MG can promote a strong antibody response to the vaccine. The pseudovirus neutralization experiment was consistent with the short-term observation results, with F-MG+RBD inducing the highest neutralizing antibody titer. The neutralizing antibodies against the original strain induced by F-MG+RBD were significantly higher than those in the RBD group (P<0.05). Figure 14 e), and also the highest mean cross-neutralizing antibody titer against the currently prevalent XBB strain. Figure 14 f). Because we observed high TNF-α expression during short-term observation, we also evaluated TNF-α expression in splenic T lymphocytes during immune recall, finding that CD4+ expression was high at this time. + The expression of TNF-α in T lymphocytes was still highest in the F-MG+RBD group, but the difference was not statistically significant. Figure 14g), while CD8 + The expression of TNF-α in T lymphocytes was still significantly higher in the F-MG+RBD group than in the MDP+RBD group (P<0.05), and also higher than in the RBD group (P=0.0719). Figure 14 h).

[0093] 6. Biocompatibility of F-MG in COVID-19 protein vaccines This invention evaluates the biocompatibility of F-MG+RBD in vivo. Based on published protocols... [2] On day 0, each mouse was injected intravenously with double the dose of F-MG (80 μg) + RBD, and acute toxicity induced by the nanofiber vaccine was assessed on day 7. Figure 15 a). Compared with control mice, mice injected with F-MG+RBD showed almost identical weight gain ( Figure 15 b). This indicates that F-MG+RBD injection had no effect on the growth of mouse body weight. Given the pyrogenicity of high-dose MDP, the rectal temperature of the mice was also observed. During the 7-day observation period, the body temperature of both F-MG+RBD mice and control mice fluctuated within the normal range for mouse body temperature, indicating that F-MG+RBD injection had no effect on mouse body temperature. Figure 15 c). In addition, the heart, liver, spleen, lungs, kidneys, and brain were harvested, and the organ coefficient (organ weight / body weight ratio) was assessed. Based on these samples, the organ coefficients showed no significant difference between mice injected with F-MG+RBD and control mice. Figure 15 (d) This indicates that the F-MG adjuvant has good biocompatibility in mice and does not accumulate in various organs to cause toxicity. At the end of the 7-day experiment, several key biochemical indicators were quantified from mouse serum samples. The expression of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in F-MG+RBD-injected mice and control mice showed no statistically significant differences compared to control mice. Figure 15 e). Immunohistochemical analysis also showed that, consistent with the control group, no inflammatory cell infiltration or organ necrosis was observed in the heart, liver, spleen, lungs, and kidneys of mice injected with F-MG+RBD. Figure 15 f). In summary, these results indicate that F-MG has good biocompatibility in mice.

[0094] This section uses self-assembled peptides as a framework to construct the nanofiber adjuvant F-MG by multivalently displaying NOD2 ligand MDP. It achieves the combination of delivery and immune activation functions as a single adjuvant and conducts long-term evaluation of its adjuvant performance in COVID-19 protein vaccines.

[0095] The research results are as follows: 1. An F-MG nanofiber adjuvant was successfully constructed and demonstrated to bind to RBD for delivery, and to promote NOD2 expression more effectively than MDP.

[0096] 2. F-MG elicited a strong humoral and cellular immune response in mice.

[0097] The humoral immune response results in mice showed that the titers of IgG and IgG subtypes in the F-MG+RBD, MDP+RBD, and RBD groups followed a consistent trend. F-MG+RBD induced the highest antibody titers, particularly eliciting higher levels of IgG2a than MDP+RBD. IgG2a has been shown to be the major antibody type mediating ADCC and CDC in mice, playing a crucial role in virus clearance, indicating that F-MG can promote a strong antibody response to the vaccine. F-MG+RBD also induced the highest neutralizing antibody titers. The pseudovirus neutralization experiment demonstrated that F-MG+RBD could produce higher neutralizing antibodies against the original strain and also cross-neutralize the currently prevalent XBB strain: live virus cross-neutralization results showed that, compared to the most widely used aluminum adjuvant, F-MG+RBD produced higher neutralizing antibody titers against the original SARS-CoV-2 strain, Delta, and Omicron strains. These results demonstrate that F-MG can promote cross-protection against the vaccine. The mouse cellular immune response results showed that, compared to mice injected with RBD, the F-MG+RBD group had higher CD4 counts. + T cells secrete more IL-4 and TNF-α:CD8 + T cells secreted more TNF-α, and the F-MG+RBD group also secreted more TNF-α compared to mice injected with MDP+RBD, indicating that F-MG can induce stronger cellular immunity. When a booster injection was given 336 days later, the CD8+ of the F-MG+RBD group showed significantly higher levels of TNF-α. + T cells still secreted the highest levels of TNF-α, and since TNF-α is related to the maintenance of plasma cell homeostasis, this supports the subsequent experimental results that F-MG has a greater advantage in sustained protection.

[0098] 3. F-MG provides long-lasting stimulation of the immune response to COVID-19 protein vaccines. Seven days after two injections, the F-MG+RBD group induced more long-lived plasma cells and memory B cells than the MDP+RBD and RBD groups, indicating that F-MG would produce more sustained antibody secretion and a higher antibody response upon re-antigen challenge, which was indeed the case. In continuous monitoring of antibody response, the F-MG+RBD group consistently showed higher antibody titers than the RBD group, and its antibody titers exhibited a slower decline trend than the MDP+RBD or RBD groups. A booster injection 336 days later also produced higher antibody and neutralizing antibody titers, demonstrating that F-MG+RBD provides long-lasting stimulation of the COVID-19 protein vaccine immune response.

[0099] 4. In vivo biosafety evaluation in mice showed that F-MG has good safety.

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nanofibrillar adjuvant, characterized in that, The chemical structural formula of the nanofiber adjuvant is: 。 2. The application of the nanofiber adjuvant according to claim 1 in the preparation of long-acting antibody-response vaccine products.

3. The application according to claim 2, characterized in that, The vaccines include novel coronavirus protein vaccines.

4. The application according to claim 2, characterized in that, The term "long-lasting" means that, compared to the initial injection of the vaccine, a second injection of the vaccine at least 336 days later produces higher antibody and neutralizing antibody titers.

5. The application according to claim 2, characterized in that, The vaccine contains the novel coronavirus RBD protein and the nanofiber adjuvant.

6. The application according to claim 5, characterized in that, The mass ratio of the novel coronavirus RBD protein to the nanofiber adjuvant is 1:2~4.

7. A novel coronavirus protein vaccine, characterized in that, The vaccine comprises the novel coronavirus RBD protein and the nanofiber adjuvant of claim 1.

8. The use of the nanofiber adjuvant according to claim 1 or the novel coronavirus protein vaccine according to claim 7 in the preparation of products that enhance the cellular immune response of the COVID-19 protein vaccine.

9. The application according to claim 8, characterized in that, The enhanced cellular immune response to the COVID-19 protein vaccine includes promoting the endosome membrane localization of NOD2.

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