CN117731767A

CN117731767A - Double-antigen display nano vaccine and preparation method and application thereof

Info

Publication number: CN117731767A
Application number: CN202311565861.XA
Authority: CN
Inventors: 刘尽尧; 陈芳洁; 张蒙蒙
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2023-11-22
Filing date: 2023-11-22
Publication date: 2024-03-22

Abstract

The invention discloses a double antigen display nano vaccine, which is a nano vaccine for wrapping the surface of nano particles after double cell membrane fusion. A method for preparing a double antigen display nano vaccine, comprising the following steps: the cell membrane expressing the virus antigen and the cell membrane expressing the tumor antigen are fused by ultrasonic to obtain a double cell membrane, and then PLGA nano particles containing an immune agonist R848 are constructed, and the double cell membrane is wrapped on the surfaces of the nano particles. The application of the double antigen display nanometer vaccine in preparing medicines for resisting tumor, resisting virus infection and enhancing immunity. The double antigen display nano vaccine can remarkably inhibit tumor growth by promoting DC cross presentation, triggering antigen specific T cell immune response and relieving Treg cell mediated immunosuppression. Immunization with these nanovaccines can significantly induce efficient production of S-specific IgG antibodies, potentially preventing related infectious complications.

Description

Double-antigen display nano vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a double-antigen display nano vaccine and a preparation method and application thereof.

Background

Cancer is an extremely complex disease, the progression and treatment of which is always accompanied by different complications. Accompanying complications, such as hypercoagulability, immunosuppression, paraneoplastic syndromes and infections, are often ignored but can lead to serious fatal consequences. Among the most common complications, infection has become a major cause of death in cancer patients. Effective strategies that can simultaneously treat cancer and protect patients from infectious complications are highly desirable.

Vaccines elicit antigen-specific immune responses by exposing mutant-derived neoantigens or pathogenic antigens to antigen presenting cells, a major pathway that mobilizes the immune system to recognize tumor cells and foreign invaders and respond. Among traditional vaccine varieties, subunit vaccines are receiving a great deal of attention for their safety, structural designability and functional adjustability. Over the past few years, various subunit vaccines have been developed that have unique characteristics such as co-delivery of molecular adjuvants, long-acting release behavior, and enhanced accumulation of targeted sites. Whereas previous methods for preparing subunit vaccines require complex and time-consuming procedures to ensure the native three-dimensional structure of the recombinant protein antigen. Encapsulation of nanoparticles with natural cell membranes is a biomimetic nanotechnology that has recently been applied to deliver a variety of intramembrane proteins. For example, encapsulation of immunoadjuvant-loaded nanoparticles with cancer cell-derived membranes can promote antigen presentation, thereby promoting tumor-specific immune responses. Therefore, the biomimetic nanotechnology provides a direct top-down approach to maintaining the correct conformation of the native antigen. Therefore, there is a need to design a double antigen display nano vaccine, and a preparation method and application thereof.

Disclosure of Invention

In order to overcome the defects in the prior art, a method for screening a microorganism group related to radiotherapy and chemotherapy is provided.

The invention is realized by the following scheme:

a double antigen display nano vaccine is characterized in that double cell membrane fusion is utilized to wrap the surfaces of nano particles.

The nanoparticle contains an immune agonist within the nanoparticle.

The nanoparticle is a PLGA nanoparticle.

The immune agonist is R848.

The cell membrane is a double cell membrane containing double antigens of tumor and virus on the surface.

The double cell membrane is 293T cell membrane for expressing spike protein S protein of novel coronavirus and B16 cell membrane for expressing tumor antigen OVA protein.

A method for preparing a double antigen display nano vaccine, comprising the following steps: the cell membrane expressing the virus antigen and the cell membrane expressing the tumor antigen are fused by ultrasonic to obtain a double cell membrane, and then PLGA nano particles containing an immune agonist R848 are constructed, and the double cell membrane is wrapped on the surfaces of the nano particles.

The specific steps of ultrasonic fusion of the cell membrane expressing the virus antigen and the cell membrane expressing the tumor antigen are as follows: extracting 293T cell membrane expressing spike protein S protein of novel coronavirus and B16 cell membrane expressing tumor antigen OVA protein, and performing ultrasonic fusion.

The double cell membranes were encapsulated on the nanoparticle surface by a liposome extruder.

The application of the double antigen display nano vaccine in preparing medicines comprises the nano vaccine which wraps the surfaces of nano particles after double cell membrane fusion, and the medicines are applied to resisting tumor, resisting virus infection and enhancing immunity.

The beneficial effects of the invention are as follows:

compared with the prior art, the invention has the following beneficial effects: the double antigen display nano vaccine can remarkably inhibit tumor growth in established and preventive tumor models by promoting DC cross presentation, triggering antigen specific T cell immune response and alleviating Treg cell mediated immunosuppression. On the other hand, immunization with these nanovaccines can significantly induce the efficient production of S-specific IgG antibodies, thereby potentially preventing SARS-CoV-2 related infectious complications. The technology provides a simple and universal platform for developing a plurality of double-mode or even multi-mode vaccines so as to synchronously treat cancer and prevent related complications, and has important research significance and clinical application value.

Drawings

FIG. 1 is a schematic representation of the use of DADNs for the treatment of cancer and the prevention of complications of infection. (A) DADNs are prepared by coating immunoadjuvant-loaded nano-particles with hybrid cell membranes, wherein the hybrid cell membranes are formed by fusing two layers of cell membranes which respectively overexpress tumor and infectious pathogenic antigens. (B) DADNs can be used as nanovaccines to simultaneously induce tumor-associated antigen-specific T cell immune responses and produce pathogenic antigen-specific IgG antibodies.

FIG. 2 is a representation of DADNs. (A and B) the (A) size and (B) surface zeta potential data of R848-NPs, SOCMVs and DADNs measured by DLS. Error bars represent standard deviation (n=3). (C) uranium acetate negatively dyes TEM images of R848-NPs and DADNs. Scale bar 100nm. (D, E) flow cytometry to detect MFI of (D) S protein and (E) egg cells on DADNs. Error bars represent standard deviation (n=3). (F) SOCMVs analyzed by tandem mass spectrometry can identify the percentage of S protein and OVA in the protein. (G and H) the percentage of other proteins from (G) mouse and (H) human cells in the protein recognizable on the hybridization membrane. Significance was assessed using Student's t-test. * P <0.01, p <0.05.

Fig. 3 is a TEM image of hybridized cell membrane vesicles negatively stained with uranium acetate. Scale bar 100nm.

FIG. 4 is a confocal image of hybridized cell membranes fused with 293T-S (red) and B16-OVA (green) membranes. Scale bar 5 μm.

FIG. 5 is a Western blot image of OVA proteins at different concentrations of free OVA and OCMVs. (B) Western blot images of S proteins of free S proteins and SCMVs at different concentrations. (C, D) (C) OVA and (D) S proteins were calibrated according to standard calibration curves for band intensities. The numbers represent estimated molecular weights in kilodaltons (kDa).

FIG. 6 shows the hydrated particle size of DADNs in PBS at 4℃at various time points.

FIG. 7 shows the viability of DC2.4 cells after incubation for 24 hours at different concentrations of DADNs. Error bars represent standard deviation (n=3).

FIG. 8 is an illustration of antigen cross presentation and T cell activation in vitro. (A) Representative CLSM images of BMDCs after 18 hours incubation with DADNs, intracellular co-localization of PLGA core (DiD, red) and hybridized cell membrane (FITC, green). Nuclei were stained with Hoechst (blue). BF stands for bright field. Scale 20. Mu.m. (B-E) (B) SIINFEKL-H-2Kb, (D) CD86, (E) CD80, and (C) PBS, SNPs, ONPs or DADNs for 18 hours. Error bars represent standard deviation (n=3). (F-I) T cell activation after 3 days of co-incubation with BMDCs incubated with PBS, SNPs, ONPs or DADNs. (F) CFSE-labeled CD8 ⁺ Representative flow cytometry histograms of T cells and (G) CFSE versus CD8 ⁺ Quantification of T cell proliferation. (H) Representative flow cytometry scatter plots and (I) tet ⁺ CD8 ⁺ Corresponding percentages of T cells. Error bars represent standard deviation (n=3). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * P<0.0001,***p<0.001,**p<0.01,*p<0.05。

FIG. 9 is SIINFEKL-H-2Kb after 18 hours incubation of BMDCs with PBS, free OVA, S protein and R848 or DADNs ⁺ And SPIKE (SPIKE) ⁺ Percentage of BMDCs. Error bars represent standard deviation (n=3). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * P<0.0001,***p<0.001,**p<0.01。

FIG. 10 shows DiD at the injection site of mice after 6 hours of treatment with PBS, diD-labeled R848-NPs or DADNs ⁺ Percent DCs. Error bars represent standard deviation (n=3). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * P<0.0001,***p<0.001,**p<0.01,*p<0.05。

FIG. 11 is a graph showing the induction of systemic immune responses. (A) Experimental design for in vivo immune response evaluation of tumor-bearing mice. Mice were subcutaneously injected 1×10 on day 0 ⁶ The B16-OVA cells were then intravenously injected PBS, SNPs, ONPs or DADNs 4 times. Mice were euthanized on day 21, spleen and TDLNs samples were collected and immune cells were collected. MFI values of (B) CD80 and (C) CD86 on (B and C) spleen DCs. (D-F) spleen-harvested CD8+ T cells (D) Ki67 ⁺ Percentage of (E) tet ⁺ CD8 ⁺ (F) IFN-gamma in T cells ⁺ Is a percentage of (c). (G) Spleen CD8 ⁺ /Treg(Foxp3 ⁺ CD25 ⁺ CD4 ⁺ ) Cell ratio. (H-J) MFI values of (H) CD80, (I) CD86 and (J) MHC-II on DCs harvested from TDLNs. (K and L) TDLNs isolated CD8 ⁺ Ki67 in T cells ⁺ Percentage (K) and CD8 ⁺ IFN-gamma in T cells ⁺ Percent (L). (M) CD8 in TDLNs ⁺ Ratio of Treg cells.

FIG. 12 shows the MFI values of SIINFEKL-H-2Kb in (A) TDLNs harvested DCs. (B) Tet in TDLNs ⁺ CD8 ⁺ Percentage of T cells. Error bars represent standard deviation (n=6). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * P<0.0001,***p<0.001,**p<0.01,*p<0.05。

FIG. 13 is a TDLNs separated CD4 ⁺ IFN-gamma in T cells ⁺ Is a percentage of (c). Error bars represent standard deviation (n=6). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * P<0.0001,***p<0.001,**p<0.01,*p<0.05。

FIG. 14 shows IL-12p40 levels in serum samples. Error bars represent standard deviation (n=6). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * p <0.05.

FIG. 15 is the activation of an intratumoral immune response. Tumor tissue sections of immunized mice, immune cells were isolated, and intratumoral immune responses were analyzed. (A, B) MFI values of (A) SIINFEKL-H-2Kb and (B) CD80 on tumor tissue DC. (C-E) (C) CD8 ⁺ T cells, (D) tet ⁺ CD8 ⁺ T cells and (E) Ki67 ⁺ The percentage of total T cells in the tumor. (F) CD8 ⁺ /TrThe corresponding proportion of eg cells. (G and H) (G) average and (H) individual tumor growth curve. (I) immunofluorescent staining images of tumor tissue of treated mice. Apoptotic cells and nuclei were labeled with TUNEL (green) and 4', 6-diamino-2-phenylindole (DAPI, blue), respectively. Scale 50 μm. Error bars represent standard deviation (n=5). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * P<0.0001,**p<0.01,*p<0.05。

FIG. 16 is H & E staining of tumor tissue of immunized mice. Scale 100 μm.

FIG. 17 is a design of experiment for (A) in vivo tumor suppression evaluation. Mice were subcutaneously injected 1×10 on day 8 ⁶ B16-OVA cells were then subcutaneously injected every 7 days PBS, SNPs, ONPs or DADNs (n=6). Mice were euthanized on day 15 and TDLNs and serum samples were collected for analysis of immune responses. (B) tumor mean growth curve. (C) photographs of tumor sections at day 15 post-injection. The dashed circles indicate that no tumor was seen. (D) CD80 in TDLNs ⁺ CD86 ⁺ Percent DC. (E) CD8 in tumor ⁺ /Treg(Foxp3 ⁺ CD25 ⁺ CD4 ⁺ ) Ratio of cells. (F) IFN-gamma levels in serum. Error bars represent standard deviation (n=6). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * P<0.01,*p<0.05。

Fig. 18 is a diagram of generation of protective immunity. (A) Experimental design for evaluation of protective immunity in a prophylactic mouse model. Mice were immunized with PBS, SNPs, ONPs or DADNs every 7 days starting on day 0, 3 times, and vaccinated 5 x 10 on day 21 ⁶ B16-OVA cells. Tumor volumes were measured every other day and serum was collected at designated time points for detection of anti-S1 IgG antibodies. (B) average tumor growth curves of mice after different treatments. (C) survival of mice after vaccination. (D) kinetics of anti-S1 IgG titres in serum after immunization. (E) day 28 serum anti-S1 IgG titer levels. (F) the ratio of IgG1/IgG2a in serum was collected on day 21. Error bars represent standard deviation (B, C: n= 7;D-F: n=5). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. Survival was analyzed using log-rank test. * P<0.0001,***p<0.001,**p<0.01,*p<0.05。

Figure 19 is tumor size of individual vaccinated mice. Error bars represent standard deviation (n=7).

FIG. 20 is the kinetics of SARS-CoV-2S1 specific IgG antibody titer in serum after immunization with PBS, SNPs, ONPs or DADNs. Error bars represent standard deviation (n=6). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * P <0.0001, < p <0.001, < p <0.05.

FIG. 21 shows (A) representative CLSM images and (B) flow cytometry analysis after incubating 293T-hACE2 cells with PsV (red) after pre-incubation of PsV with serum samples of immunized mice. Nuclei were stained with Hoechst (blue). Scale 10 μm. Error bars represent standard deviation (n=3). Significance was assessed using one-way analysis of variance and Dunnett's post-hoc test. * p <0.05.

Detailed Description

The preferred embodiments of the present invention are further described below:

The nanoparticle contains an immune agonist within the nanoparticle.

The nanoparticle is a PLGA nanoparticle.

The immune agonist is R848.

Infection is one of the most common complications and is the leading cause of death in cancer patients. However, therapeutic strategies that can simultaneously inhibit tumors and protect patients from infection have been rarely reported. The present Dual Antigen Display Nanovaccines (DADNs) can elicit synergistic immune activation for the treatment of cancer and the prevention of infectious complications. The preparation method is to wrap the nano-particles loaded with the immune adjuvant by a mixed coating, and the mixed coating is formed by fusing cell membranes which are respectively genetically engineered to express tumor and infectious pathogen antigens. Due to the presence of the double antigen combination, DADNs are able to promote maturation of dendritic cells, and more importantly trigger cross presentation of the two combined antigens. In vivo studies, DADNs can reverse immunosuppression by stimulating tumor-associated antigen-specific T cell responses, thereby significantly delaying tumor growth in mice. In a prophylactic study, these nanovaccines can also elicit potent protective immunity against tumor challenge and elicit the production of pathogenic antigen-specific antibodies. This work provides a unique approach to the development of bimodal vaccines, with the hope of simultaneously treating cancer and preventing infection.

Preferred embodiments of the present invention will be further described with reference to the accompanying drawings:

1. materials and methods:

1.1 preparation of cell membranes

First, spike protein is transduced onto HEK-293T cells to prepare genetically engineered 293T-S cells. Human 293T and B16-OVA cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin streptomycin (Gibco). Cells in the presence of 5% CO ₂ Is humidified at 37 DEG CCulturing in a incubator. 293T cells were harvested after incubation in T175 flasks until confluence was sufficient. Cells were washed 3 times with 1 Xphosphate buffered saline (PBS) (800 Xg centrifugation), suspended in hypotonic lysis buffer containing 30mM Tris HCl (pH 7.5,Sigma Aldrich), 225mM D-mannitol (Sigma Aldrich), 75mM sucrose (Sigma Aldrich), 0.2mM ethylene glycol-bis (-aminoethyl ether) - (N, N, N ', N' -tetraacetic acid (EGTA, sigma Aldrich) and protease and phosphatase inhibitors (Thermo Fisher Scientific.) to quantify the concentration of 293T-S and B16-OVA membrane proteins, 293T-S and B16-OVA membranes were added to B16-OVA membranes at a 1:1 protein ratio using BCA protein kit (Beyotime Biotechnology), and the hybridized membranes were stained with fitc-labeled anti-ovalbumin (OVA, abcam) and SARS-CoV-2 (2019-nCoV) spike S1 antibodies (fluorescent) and then stained with a cross-reader-chip anti-IgG (TSC) and subsequently cross-chip anti-light-adsorption (anti-IgG) were performed on the cross-chip (cross-chip) membrane by using a laser microscope (FIG. 546).

1.2 preparation of Nanoparticles (NPs)

PLGA nanoparticles were prepared using the RG502 PLGA polymer (EVONIK, 0.16-0.24 dL/g) by the nanoprecipitation method. First, 5mg of PLGA polymer and 250 μ g R848 (10 mg/mL) were dissolved in 3mL of Dichloromethane (DCM), and the prepared mixture was dropped into 15mL of 0.2% polyvinyl alcohol and stirred in air at room temperature for 12h (500 rpm). After stirring, NPs were centrifuged at 10000×g for 10 minutes, washed 3 times with distilled water, and then lyophilized. The NPs were dissolved in acetonitrile and the release amount of R848 was 35. Mu.g as determined by ultraviolet-visible spectrophotometry. The drug loading content and encapsulation efficiency of R848 were calculated as follows.

Load content= (mass of load R848/mass of nanoparticle) ×100%

Packaging efficiency= (mass loaded into R848/total mass loaded into R848) ×100%

1.3 preparation of DADNs

NPs solution (0.4 mL,10 mg/mL) was added to 293T-S membrane solution (0.4 mL,20 mg/mL) or B16-OVA membrane solution (0.4 mL,20 mg/mL), respectively. NPs solution (0.4 mL,20 mg/mL) was mixed with the mixed film solution (0.4 mL,40 mg/mL) and extruded on a liposome extruder (Avanti Polar Lipids) at 400nm and 200nm, respectively, to give DADNs. Excess membrane was removed by centrifugation at 10000rpm/min for 5 min. Finally, the resulting DADNs were resuspended with 1×pbs. The DADNs were divided into two groups and antigen stained to confirm successful hybridization. OVA protein staining was performed with fitc-labeled anti-OVA (Abcam). S protein staining was performed with rabbit anti-SARS-CoV-2 (2019-nCoV) Spike S1 MAb (sinobological) and fitc conjugated goat anti-rabbit IgG (H+L) (Abcam), flow cytometry analysis. A fitc-labeled mouse IgG1 isotype (eBioscience, P3.6.2.8.1) was used as a control. The size and surface zeta potential of NPs and DADNs were measured using a Malvern ZEN 3600Zetasizer dynamic light scattering method. Morphology was observed with a transmission electron microscope (TEM, hitachi HT7700, tokyo, japan).

Cytotoxicity of 1.4DADNs

To assess cytotoxicity of DADNs, 2×10 was used ⁴ DC2.4 cells (dendritic cell line) were seeded in 96-well plates and incubated overnight at 37 ℃. DADNs prepared at various concentrations of 0.025-1.6 mg/mL were added to the plate, incubated at 37℃for 24h, 10. Mu. LCCK-8 solution (Beyotidme, china) was added to each well, incubated at 37℃for 1h, and absorbance at 450nm was recorded using a microplate reader (BioTek, USA).

1.5 antigen Cross presentation of BMDCs

To obtain bone marrow-derived dendritic cells (BMDC), isolated bone marrow cells were cultured in RPMI 1640 containing GM-CSF (10 ng/mL, peprotech) and IL-4 (10 ng/mL, peprotech) for 6 days. Non-adherent cells and loosely adherent cells were collected, stained with anti-CD 11c antibody, and the differentiation efficiency of BMDCs was verified. BMDCs (1X 10) ⁶ ) Detection was collected after 18h incubation with DADNs (16 mg/mL), SNPs (8 mg/mL) or ONPs (8 mg/mL). Flow cytometry (CytoFLEX, beckman Coulter) analyzed anti-mouse APC H-2Kb, anti-mouse CD86 (B7-2) -APC (Biolegend, clone GL-1) and anti-mouse CD80-PE (eBioscience, clone16-10A 1) bound by SIINFEKL antibody (Biolegend, clone 25-D1.16). In addition, the treated BMDCs were stained with SARS-CoV-2 spike receptor binding domain antibody (SinoBiological), followed by staining with a fitc-labeled goat anti-rabbit IgG (H+L) secondary antibody (SinoBiological). To detect the uptake of DADNs by BMDCs, cells (1X 10) ⁶ ) Incubation with DADNs (16 mg/mL) wherein PLGA and impuritiesThe cross cell membranes were labeled with 1,1 '-octacosyl-3, 3' -tetramethylindole dicarboncyanine (DiD, beyotime Biotechnology) and fitc-labeled anti-ova antibody, respectively, and incubated at 37℃for 18h. After staining with Hoechst 33342, BMDCs were observed with CLSM.

1.6 in vitro cross-presentation of T cells

According to CD8 ⁺ T cell isolation MACS kit (Miltenyi Biotec) isolation of primary CD8 ⁺ T cells were then stained with carboxyfluorescein succinimidyl ester (CFSE, eBioscience). Subsequently, the cells were diluted to 2×10 ⁶ Each mL was placed in a 12-well cell culture plate, and the degree of proliferation of T cells was evaluated. DADNs, ONPs or SNPs were co-cultured with BMDCs, which were then incubated with T cells at a ratio of 10:1 for 3 days at 37 ℃. After incubation, T cells were collected and analyzed by flow cytometry. To assess antigen-specific presentation, T cells were collected and stained with H-2Kb OVA Tetramer-SIINFEKL-APC (MBL International). With Zombie NIR ^TM Fixable Viability Kit (Biolegend, 423105) dead cells were removed. Flow cytometry calculated the percent OVA-specific T cells.

1.7 ability of DADNs to target DCs in vivo

To test the ability of DADNs to target DCs in vivo, mice were subcutaneously injected with 100. Mu.L of PBS, di-labeled R848-NPs (10 mg/mL), and DADNs (10 mg/mL), respectively. Skin tissue was harvested 6h after injection and lymphocytes were isolated using lymphocyte isolation medium (Beyotime Biotechnology). Flow cytometry detection of CD11c ⁺ DiD in cells ⁺ Is a ratio of (2).

1.8 tumor-specific immunity elicited by DADNs in vivo

Female C57BL/6 mice of 6-8 weeks old were inoculated subcutaneously 1X 10 on day 0 ⁶ Individual B16-OVA cells (right hind leg). Mice were inoculated on day 13 (tumor size reached 50-100 mm) ³ ) Initially, 100. Mu.L of PBS, SNPs (15 mg/mL), ONPs (15 mg/mL) or DADNs (30 mg/mL) were administered daily via the tail vein for 4 days. Tumor volumes were measured every other day with digital calipers and calculated to be 0.5 x length x width ² .5 days after the fourth injection, spleen, lymph node and tumor tissues were collected and analyzed for CD8 by flow cytometry ⁺ CD3 ⁺ 、CD3 ⁺ Ki67 in (A) ⁺ 、CD3 ⁺ IFN-gamma of (B) ⁺ Ratio of ova-tetramer, SIINFEKL-H-2Kb, CD80 and CD86 at CD11c ⁺ Average fluorescence intensity on cells and Treg cells. Cytokine levels were detected by enzyme-linked immunosorbent assay (ELISA), and serum samples were isolated from peripheral blood. Excision of tumor tissue, H&E and TUNEL immunofluorescent staining.

1.9 antiviral and antitumor vaccination

To investigate the protective effect of vaccination, 6-8 week old C57BL/6J female mice were subcutaneously vaccinated with 200. Mu.L of SNPs (30 mg/mL), ONPs (30 mg/mL) and DADNs (60 mg/mL) on days 0, 7 and 14, respectively. On day 21, mice were subcutaneously injected 5×10 on the right side ⁶ B16-OVA cells. Tumor volume was measured every other day, once tumor size exceeded 2000mm ³ Mice were euthanized. Serum was isolated from peripheral blood on days 0, 14, 21, 28, respectively.

1.10ELISA

Recombinant SARS-CoV-2S1 protein (2. Mu.g/mL) was diluted with bicarbonate buffer (pH 9.6) and plated onto 96-well microplates (100. Mu.L/well) overnight at 4 ℃. Then blocked with 2% Bovine Serum Albumin (BSA) in wash (1 XPBS buffer with 0.5% Tween-20 (v/v)) for 2 hours at 37 ℃. After washing the microwell plates three times with buffer, serum was serially diluted twice in PBS containing 1% bsa and incubated for 1 hour at 37 ℃. The microwell plates were washed three times with buffer. Goat anti-mouse IgG (HRP) (1:10 universal dilution, ab 6789) was used and incubated for 1h at 37 ℃. Color development was performed with 100. Mu.L of 3,3', 5' -tetramethylbenzidine (TMB, solarbio). Finally, stop solution (1M H) ₂ SO ₄ ) The reaction was stopped. Absorbance was measured with a microplate reader at 450 nm. To confirm the type of antibody reaction, serum isolated on day 21 of vaccinated mice was added separately, and after washing 3 times with buffer, goat anti-mouse IgG1 (HRP) (1:10000 dilution, ab 97240) and goat anti-mouse IgG2a (HRP) (1:10000 dilution, ab 97245) were used, respectively. To detect the levels of pro-inflammatory cytokines, a mouse IL-12p40ELISA kit (Biolegend) was used.

1.11 flow cytometric analysis

DC maturation and antigen presentation was detected by fluorescent conjugated antibody staining, including anti-mouse CD11c-FITC (bioleged, clone N418), anti-mouse CD11b-PerCP/Cy5.5 (bioleged, clone M1/70), anti-mouse H-2Kb binding SIINFEKL-APC (bioleged, clone 25-D1.16), and anti-mouse CD80-PE (eBioscience, clone16-10A 1). To analyze immune cell subsets and inflammatory cytokines in spleen, lymph nodes and tumor tissues, single cell suspensions prepared from these samples were examined using flow cytometry. The primary antibodies used included anti-mouse CD3-PerCP/Cyanine 5.5 (Biolegend, clone145-2C 11), anti-mouse CD8-PE (eBioscience, clone 53-6.7), anti-mouse CD8a-FITC (eBioscience, clone 53-6.7), anti-mouse CD16/32 (Biolegend, clone 93), T-select H-2Kb OVA tetramer-SIINFEKL-APC (MBL), anti-mouse CD4-APC (eBioscience, clone RM 4-5), anti-mouse CD25-APC (eBioscience, clone PC 61.5), anti-mouse CD45-PE/Cyanine7 (Biolegend, clone 30-F11), anti-mouse CD86 (B7-2) -APC (Biolegend, GL-1). After incubation on ice for 30 min, cells were washed, fixed and stained intracellular with Foxp 3/transcription factor fixation/permeation kit (Invitrogen). Intracellular cytokines or nucleoproteins were determined by staining with anti-mouse Ki67-FITC (biolgend, clone SolA 15), anti-mouse IFN-. Gamma. -PE/Cyanine7 (biolgend, clone XMG 1.2) or anti-foxp 3-PE (biolgend, clone MF-14). Data were acquired on a CytoFLEX flow cytometer and analyzed using CytExpert (Beckman Coulter) and FlowJo (Tree Star) software.

1.12 immunoblotting

And detecting the contents of OVA and S proteins on the DADNs by using a western blot method. 293T-S or B16-OVA cell membrane-derived vesicles (called SCMVs or OCMVs) were lysed with RIPA buffer (Beyotime, china) to extract total proteins. After centrifugation at 4500 Xg for 5min, the supernatant was collected, mixed with loading buffer, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred through a polyvinylidene fluoride (PVDF) membrane. PVDF membranes were incubated with anti-ova antibodies (1:40000, PA5-97525, invitrogen) or anti-S antibodies (1:10000, A20136, abclonal) and the corresponding secondary antibodies (1:10000, AS014, abclonal). Protein bands were detected using an Amersham Imager 680 blot and gel Imager (GE Analytical Instruments, USA). Image J is used to quantify the intensity of the band. Standard curves for OVA or S proteins were calculated from their band intensities.

1.13 inhibition of SARS-CoV-2PsV infection by DADNs

A serum sample of the mice was collected on day 15 after immunization, mixed with 50. Mu.L of pseudo-SARS-CoV-2 (PsV, p 240.5. Mu.g/mL) at a volume ratio of 1:1 for 1h at 37℃and then stained with Cy5.5-NHS for PsV. PsV infection was assessed using a 293T cell line (293T-hACE 2) stably expressing human ACE 2. Adding 5X 10 ⁴ The 293T-hACE2 cells were incubated at 37℃for 24 hours, washed with PBS, and examined for infection of 293T cells by confocal microscopy and flow cytometry at PsV.

1.14 statistical analysis

Data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0. Both sets of comparisons were tested using unpaired Student's t. Multiple comparisons were made using the Dunnett post-test one-factor anova and the Bonferroni post-test two-factor anova. Survival was analyzed using log-rank test. Significance is defined as P <0.05 (< 0.05, <0.01, <0.001, < 0.0001).

2. Results

2.1 preparation and characterization of the nanovaccine

To generate cell membranes displaying the S protein, we first established an S protein over-expression HEK 293T (293T-S) cell line. The S protein contains a receptor binding domain through which SARS-CoV-2 binds to and enters the cell. B16 melanoma cells overexpressed OVA model tumor antigens by genetic engineering. Extracting 293T-S and B16-OVA cell membranes, and performing ultrasonic fusion to obtain a hybridization membrane. R848 is an immunoadjuvant of TLR 7/8, triggering an immune response of T helper (Th) 1 to tumor and viral infections, encapsulated in PLGA nanoparticle cores by a double emulsion process. At a drug administration concentration of 250 μg, the loading rate of R848 was calculated to be 0.7% and the encapsulation efficiency was 14%. The obtained hybrid cell membrane was coated on the R848-loaded PLGA nanoparticles (R848-NPs) by repeated extrusion.

Dynamic Light Scattering (DLS) data showed that the average hydrodynamic diameter slightly increased from 133.5±1.1nm to 140.8±0.6nm after the hybrid film was applied (fig. 2A). Surface zeta potential measurements showed that nanoparticles (DADNs) displaying S and OVA antigens were about 13mV less negatively charged than PLGA nanoparticles, but comparable to hybrid cell membrane derived vesicles (SOCMVs) (fig. 2B). The size of the negative-stained DADNs was confirmed by Transmission Electron Microscopy (TEM) and showed typical core-shell spherical nanostructures (fig. 2C and 3). To confirm fusion of these two cell membranes, we labeled SOCMVs with antibodies specific for S and OVA proteins. Images of Confocal Laser Scanning Microscopy (CLSM) demonstrated the presence of signature of 293T-S and B16-OVA cell membranes by SOCMVs (fig. 4). In addition, flow cytometric analysis showed simultaneous expression of S1 and OVA proteins on DADNs (fig. 2D and E) compared to the corresponding isotype control, further confirming adequate membrane fusion and retention of intact antigen on the hybridized cell membrane. Calculated, 30 μg DADNs carry 67.71ng and 0.24ng of OVA and S proteins, respectively (FIG. 5). And comprehensively analyzing the protein components of the hybridization membrane by adopting a tandem mass spectrometry method. Of the identifiable peptides, the percentage of peptides belonging to the S and OVA proteins was about 6.66% and 7.77%, respectively (fig. 2F). The fusion membrane contained other abundant peptides from mouse and human species (fig. 2G and H). In addition, in Phosphate Buffered Saline (PBS) at 4 ℃, the size change of DADNs over 4 weeks was negligible, indicating satisfactory storage stability (fig. 6). Importantly, even if the DADNs concentration was increased to 1.6mg/mL, no cytotoxicity was observed in vitro (fig. 7), indicating good cell compatibility. Taken together, these results demonstrate the successful preparation of DADNs that display both tumor and viral antigens.

2.2 in vitro dendritic cell antigen cross presentation

In view of the key role of endocytosis in DC antigen processing and presentation, cellular uptake of DADNs was first assessed using Fluorescein Isothiocyanate (FITC) -labeled hybridization membranes and 1,1 '-octacosyl-3, 3' -tetramethylindole dicarbocyanine (di) -stained PLGA. CLSM imaging showed that bone marrow derived DC (BMDC) fluorescent signals were evident after 18 hours of incubation, indicating rapid cellular internalization (fig. 8A). Co-localization of fluorescent signals associated with PLGA nuclei and hybridized membranes further confirmed the entire structure of DADNs in BMDCs, indicating that both antigen and R848 agonist were taken up. After confirmation of effective cellular uptake, BMDCs were tested for antigen cross presentation. PBS, 293T-S cell membrane coated nanoparticles (SNPs) and B16-OVA cell membrane coated nanoparticles (ONPs) were used as controls, respectively. After 18 hours of incubation, flow cytometry was performed to detect OVA-specific H-2 Kb-restricted peptides and anti-SIINFEKL-H-2 Kb antibodies. The Median Fluorescence Intensity (MFI) of SIINFEKL-H-2Kb of ONPs treated BMDCs was increased 2.1-fold and 1.7-fold compared to PBS and SNPs groups (FIG. 8B), demonstrating that antigen cross-presentation by OVA was promoted. In addition, SNPs treatment induced 2.4-fold and 1.3-fold increases in spike+bmdcs percentage over PBS and ONPs groups (fig. 8C). Treatment with DADNs showed the highest increase in MFI of SIINFEKL-H-2Kb and spike+bmdcs percentages on BMDCs, making the double antigen demonstrate important evidence that hybrid nanovaccines synergistically enhance antigen cross presentation. Furthermore, MFI of co-stimulatory molecule CD86 on BMDCs was significantly increased following SNPs or ONPs treatment compared to PBS group (fig. 8D). Maturation of DCs can be explained by the incorporation of R848, R848 being an effective molecular adjuvant, acting on active DCs through the myeloid differentiation factor dependent pathway. Also, as a result of synergy, DADNs induced the most significant increase in CD80 expression on BMDCs (fig. 8E). Furthermore, the percentage of SIINFEKL-H-2kb+ and spike+bmdcs in the DADNs group was still increased compared to the combination of free OVA, S protein and R848 (fig. 9), highlighting the great potential for antigen presentation.

To assess whether DADNs can promote antigen-specific immune responses, we examined CD8 ⁺ Proliferation of T cells, CD8 ⁺ T cells are critical for tumor suppression and virus elimination. BMDCs collected after incubation of DADNs, and CD8 ⁺ T cells were co-incubated for 3 days. Detection of CD8 by carboxyfluorescein succinimidyl ester (CFSE) ⁺ Proliferation of T cells, the activation ability of DADNs was evaluated. As shown in fig. 8F and G, all BMDCs incubated with ONPs, SNPs, and DADNs triggered higher cd8+ T cell proliferation compared to PBS group, with highest proliferation for SNPs and DADNs groups. Furthermore, SIINFEKL-MHC-I tetramers in ONPs group compared to PBS and SNPs group ⁺ CD8 ⁺ (tet ⁺ CD8 ⁺ ) The percentage of T cells increased significantly (fig. 8H and I). Although SARS-CoV-2 has been reported to limit antigen presentation by down-regulating MHC-I and MHC-II molecules of DC and subsequently inhibit T cell cross-reactivity, it is comparable to the ONPs groupIn contrast, the DADNs group observed tet ⁺ CD8 ⁺ T cell levels are elevated. This increase suggests that the introduction of S protein has limited negative impact on immune activation and that the two antigens produce a synergistic effect by membrane fusion binding. In general, DADNs can promote maturation of DCs and initiate antigen-specific immune responses.

2.3 eliciting a systemic anti-tumor immune response

The targeting ability of DADNs to DCs was next evaluated in vivo. Mice were subcutaneously injected with PBS, di-labeled R848-NPs, and DADNs, respectively. Flow cytometry analysis demonstrated that the hybrid membrane coating greatly increased internalization of R848-NPs by DCs (fig. 10), mainly due to easier binding between the hybrid membrane and DCs. This result suggests that DADNs have the potential to enhance immune responses in vivo. After demonstrating in vitro antigen cross-presentation and cross-priming effects, the ability of DADNs to elicit systemic anti-tumor immune responses was assessed using a B16-OVA melanoma mouse model. 5 days after the fourth dose of vaccination, tumor-bearing mice were euthanized and samples were collected to detect immune responses (fig. 11A). Since the spleen is the primary secondary lymphoid organ that captures pathogens and related antigens in the blood, the immune response of the spleen after immunization with DADNs was examined. Flow cytometric analysis showed significant up-regulation of CD80 and CD86 co-stimulatory markers on DCs after inoculation with dadnas, consistent with the results of in vitro experiments (fig. 11B and C). Mice treated with DADNs stimulated the highest levels of Ki67 expression, suggesting that binding of tumor and viral antigens may promote proliferation of cd8+ T cells (fig. 11D). The ability of DADNs to induce antigen-specific T cell immune responses was then examined. As shown in FIG. 11E, the ONPs group and the DADNs group tets ⁺ CD8 ⁺ A considerable increase in the percentage of T cells occurs. Only DADNs significantly improved CD8 ⁺ Interferon gamma (IFN-gamma) levels produced by T cells (fig. 11F). Thus, it is speculated that DADNs may mediate an effective anti-tumor response because the cytokine IFN-gamma is CD8 ⁺ An indicator of T cell toxicity. Furthermore, foxp3 ⁺ CD25 ⁺ CD4 ⁺ Regulatory T (Treg) cells express immunosuppressive receptors, produce large amounts of immunosuppressive cytokines, suppress tumor-specific immune responses, and promote tumor immune evasion. Among other thingsIn comparison to the group, DADNs-immunized mice CD8 ⁺ The Treg cell fraction was significantly increased, indicating a reversal of immunosuppression (fig. 11G).

Tumor draining lymph nodes (tumor-drain lymph nodes, TDLNs) play a key role in eliciting potent anti-tumor immunity. Next, antigen-specific immune responses induced by DADNs in TDLNs were studied. Consistent with the results obtained in the spleen, treatment with DADNs triggered the highest levels of SIINFEKL peptide on DCs (fig. 12A), and the highest upregulation of costimulatory molecules including CD80, CD86, and MHC-II (fig. 11H-J). More importantly, mice treated with DADNs obtained the highest percentage of tet ⁺ CD8 ⁺ T cells, which demonstrated stimulation of the most potent antigen-specific T cell immune response in all treatment groups (fig. 12B). T cells of the DADNs group also showed the most abundant proliferative capacity and IFN-gamma secretion capacity (FIG. 11K, L). Furthermore, only treatment with DADNs triggered an increase in IFN- γ production in cd4+ T helper cells (fig. 13). TDLNs have been reported to constitute a unique immune environment that creates systemic immune tolerance by enhancing effector T cell energy and stimulating Treg cell activation. As expected, immunization with dadnas reversed the local environment from immune tolerance to immune activation, as evidenced by an increase in the cd8+/Treg cell ratio of the dadnas group (fig. 11M). Meanwhile, proinflammatory cytokines, especially interleukin-12 (IL-12), can destroy the functions of Treg cells and promote anti-tumor immunity. Consistent with this concept, IL-12 levels in serum of the DADNs group mice remained highest in all treatment groups (fig. 14). In short, these data reveal that DADNs can elicit potent systemic anti-tumor immune responses in tumor-bearing mice.

2.4 Induction of an intratumoral immune response

Given that the tumor microenvironment is critical for tumor progression and immune evasion, the intratumoral immune response that occurs after vaccination was detected. Likewise, treatment with DADNs significantly enhanced OVA presentation of DCs and co-stimulatory signaling of CD80 within the tumor (fig. 15A and B). At the same time, the mixed nanometer vaccine effectively promotes CD8 ⁺ T cell specific antigen specific tet ⁺ CD8 ⁺ Infiltration of T cells into tumors (FIGS. 15C and D) and tumor infiltration of T cellsProliferation (FIG. 15E). Thus, DADNs can drive CD8 ⁺ T cells directly recognize and kill tumor cells. The increase in the cd8+/Treg cell ratio in tumor tissue of DADNs treated mice further demonstrated successful alleviation of the immunosuppressive tumor environment (fig. 15F). Tumor volumes were monitored every other day after vaccination to evaluate the anti-tumor response of DADNs activation. The tumor growth of the DADNs-injected mice was significantly retarded compared to PBS, SNPs, and ONPs treatments (fig. 15G and H). Furthermore, tumor growth delay was mainly due to DADNs-induced apoptosis (fig. 15I), which was further confirmed by the apparent damage of hematoxylin and eosin stained tumor tissue as demonstrated by tumor section end deoxynucleotidyl transferase dUTP notch end labeling (TUNEL) experiments (fig. 16). Cytotoxic CD8 was reported ⁺ T cells are responsible for killing tumor cells, and helper T cells maintain the function of cytotoxic T cells. Dual antigens can activate a variety of immune cell types, including cytotoxic and helper T cells, which can synergistically elicit an effective immune response. Then, tumor inhibition by DADNs was again confirmed by treating tumor-bearing mice with the nanovaccine every 7 days (fig. 17A). Treatment with DADNs significantly delayed the increase in tumor volume compared to all other groups (fig. 17B and C), while mature DCs in TDLNs (CD 80 ⁺ CD86 ⁺ ) Up-regulation of the percentage of tumor sites CD8 ⁺ The proportion of Treg cells was increased and the level of IFN- γ was increased in the serum 1 day after the last immunization (fig. 17D-F). Thus, DADNs reverse the immunosuppressive tumor microenvironment by promoting intratumoral antigen-specific T cell immunity and triggering tumor cell apoptosis.

2.5 protective immunity is generated

To determine whether DADNs were able to generate protective immunity against tumors and infectious pathogens, we established a prophylactic mouse model (fig. 18A). Three doses of DADNs were injected on days 0, 7 and 14, respectively, and mice were challenged with B16-OVA melanoma cells on day 21. As shown in fig. 18B and 19, both the DADNs group and the ONPs group exhibited significant tumor growth inhibition compared to the other treatment groups. In the ONPs and DADNs pre-treated groups, only 1 mouse showed tumor growth, and the remaining mice were completely resistant to tumor challenge, with no tumor development found during the 34 day observation period. The survival curves of the animals also reflect the generation of strong protective immunity, with median survival times of 22 days and 20 days for PBS and SNPs groups of mice, respectively (fig. 18C). In contrast, each group vaccinated with DADNs and ONPs developed only one tumor mass on day 28 and day 30, respectively. The protective effects of ONPs and DADNs are associated with the binding of tumor antigens and immunoadjuvants.

Next, we assessed the ability of DADNs to induce the production of anti-S protein IgG antibodies, which play a key role in protecting the body from SARS-CoV-2 invasion. As shown in fig. 18D and 20, the anti-s IgG antibody titer in mice after inoculation with DADNs significantly increased on day 14 compared to all control groups. More importantly, the extent of improvement in anti-S protein IgG antibody production was greatly enhanced over time to 21 days. Even when the time was prolonged to 28 days, a significant increase in antibody levels could be detected in serum samples collected from mice immunized with DADNs compared to the ONPs group (fig. 18E). The ratio of IgG1/IgG2a is considered an indicator of Th1/Th2 polarization. The DADNs induced a significant decrease in the IgG1/IgG2a ratio, indicating that Th1 immune response was induced, which is critical for preventing infection (fig. 18F). Subsequently, we also investigated the ability of DADNs-induced anti-S protein IgG antibodies to prevent viral infection. Whereas angiotensin converting enzyme II (angiotensin converting enzyme II, ACE 2) on the cell surface is the main target for SARS-CoV-2 invasion, the human 293T cell line (293T-hACE 2) expressing ACE2 was used to assess the infection status of pseudotyped SARS-CoV-2 (PsV). Both CLSM imaging and flow cytometry analysis showed that DADNs immunized mouse serum was effective in preventing PsV from invading 293T-hACE2 cells (fig. 21) compared to PBS group, indicating that DADNs immunized mouse serum could produce protective anti-S protein IgG antibodies against PsV. The above results indicate that the hybrid nanovaccine can activate strong protective immunity by eliciting an effective anti-tumor immune response and an effective antigen-specific IgG antibody response. The invention obtains a nanometer vaccine medicine capable of effectively treating tumor and preventing infection by triggering synergistic immune activation for treating cancer and preventing infection complications.

While the invention has been described and illustrated in considerable detail, it should be understood that modifications and equivalents to the above-described embodiments will become apparent to those skilled in the art, and that such modifications and improvements may be made without departing from the spirit of the invention.

Claims

1. A double antigen display nanovaccine, characterized in that: the nano vaccine is a nano vaccine which wraps the surface of the nano particle after double cell membrane fusion.

2. The double antigen display nanovaccine of claim 1, wherein: the nanoparticle contains an immune agonist within the nanoparticle.

3. The double antigen display nanovaccine of claim 2, wherein: the nanoparticle is a PLGA nanoparticle.

4. The double antigen display nanovaccine of claim 2, wherein: the immune agonist is R848.

5. The double antigen display nanovaccine of claim 1, wherein: the cell membrane is a double cell membrane containing double antigens of tumor and virus on the surface.

6. The double antigen display nanovaccine of claim 5, wherein: the double cell membrane is 293T cell membrane for expressing spike protein S protein of novel coronavirus and B16 cell membrane for expressing tumor antigen OVA protein.

7. A method of preparing a double antigen display nanovaccine as claimed in claims 1-6, comprising the steps of: the cell membrane expressing the virus antigen and the cell membrane expressing the tumor antigen are fused by ultrasonic to obtain a double cell membrane, and then PLGA nano particles containing an immune agonist R848 are constructed, and the double cell membrane is wrapped on the surfaces of the nano particles.

8. The method for preparing the double antigen display nano vaccine according to claim 7, wherein the specific steps of ultrasonic fusion of the cell membrane expressing the virus antigen and the cell membrane expressing the tumor antigen are as follows: extracting 293T cell membrane expressing spike protein S protein of novel coronavirus and B16 cell membrane expressing tumor antigen OVA protein, and performing ultrasonic fusion.

9. The method for preparing the double antigen display nano vaccine according to claim 7, wherein the method comprises the following steps: the double cell membranes were encapsulated on the nanoparticle surface by a liposome extruder.

10. Use of a double antigen display nanovaccine as claimed in claims 1-6 for the preparation of a medicament, characterized in that: the prepared medicine contains the nano vaccine which wraps the surface of the nano particle after double cell membrane fusion, and the medicine is applied to resisting tumor, resisting virus infection and enhancing immunity.