Preparation method and application of high-efficiency targeted swine fever nano vaccine

Abstract

The invention relates to molecular biology and biomedicine technologies, and aims to provide a preparation method and application of an efficient targeting swine fever nano vaccine. The invention provides an APC cell targeted hog cholera virus self-assembled protein nanoparticle, which is obtained by carrying out fusion connection expression on a recombinant plasmid of an APC cell targeted nanoparticle mi3-PTC with a sequence as shown in SEQ ID NO.3 and a hog cholera virus antigen with a sequence as shown in SEQ ID NO.5-12, and further mixing with pharmaceutically acceptable auxiliary materials to prepare a vaccine product for preventing and / or treating hog cholera virus infection. The APC cell targeting polypeptide can be displayed on the surface of the nanoparticle mi3, and the nanoparticle is directionally carried to the APC cell expressing CD163, so that the internalization, storage and distribution capabilities of the nanoparticle are remarkably improved in vivo and in vitro, and the vaccine can remarkably improve the immune response to the swine fever antigen.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and biomedical technology, and relates to a method for preparing a highly efficient and targeted swine fever nano vaccine and its application. Background Art

[0002] Classical swine fever (CSFV) is a highly lethal and contagious infectious disease caused by the classical swine fever virus (CSFV). It primarily affects domestic and wild boars, causing significant economic losses to the swine industry worldwide. The attenuated live vaccine strain C is currently the primary vaccine for CSFV control, but it lacks the ability to differentiate infected from vaccinated animals (DIVA) and carries potential biosafety risks. Therefore, CSFV E2 subunit vaccines, which possess DIVA properties, are considered a viable option for controlling and eradicating CSFV. However, due to the relatively weak immunogenicity of the single antigen in subunit vaccines and their inability to induce cellular immunity, they may not provide sufficient immune protection. Therefore, there is an urgent need to develop safer, more economical, and more effective CSFV subunit vaccines to overcome the efficacy bottlenecks and development strategy limitations of subunit vaccines.

[0003] Mi3 is a self-assembling protein. Sixty mi3 subunits form nanoparticles with a structure similar to that of a virus-like peptide (VLP). Their surface displays exogenous epitopes or proteins in an orderly and repetitive manner. The spatial arrangement of exogenous antigens resembles pathogen-associated molecular patterns (PAMPs) found on natural pathogens, enhancing the cross-linking reaction between antigens and BCRs and activating both cellular and humoral immunity, thereby inducing a stronger immune response than subunit vaccines. Currently, nanoparticle vaccines still suffer from nonspecific distribution, which can easily trigger adverse reactions, and most have difficulty reaching antigen-presenting cells (APCs) effectively.

[0004] CD163 is a typical type I glycosylated protein belonging to the cysteine-rich scavenger receptor family. It is primarily expressed in monocyte / macrophage lineages, such as splenic dendritic cells, alveolar macrophages, and liver Kupffer cells, and is also expressed in African green monkey kidney cells (MA-104, Marc-145, and CL2621). CD163 acts as a pattern recognition receptor, such as the erythroblast adhesion receptor, the tumor necrosis factor-like weak inducer of apoptosis (TWEAK), and receptors for diverse pathogens, including bacteria and viruses, and can bind to a wide range of ligands. The interaction of CD163 with its ligand can result in receptor-mediated endocytosis or trigger a signaling cascade leading to the secretion of signaling molecules. The cell-restricted expression pattern and internalization potential of CD163 make it a strong candidate for APC-targeted (immuno)therapy.

[0005] Currently reported APC targeting strategies primarily include antibody-mediated targeting and APC-targeting peptide-mediated targeting. Antibody-mediated APC targeting strategies include monoclonal antibody (mAb) and single-chain antibody (SCAb) targeting. Fusion expression of exogenous antigens with APC-targeting antibodies enhances APCs' ability to process exogenous antigens, thereby increasing the antigen's immunogenicity. Compared to monoclonal and SCAbs, nanobodies possess unique advantages in targeting affinity and delivery effectiveness due to their small molecular weight, strong permeability, and longer CDR3 regions.

[0006] The present invention proposes the preparation and application of a highly efficient targeted swine fever nanovaccine based on the CDR3 targeting peptide of the CD163 nanoantibody. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a method for preparing and applying a highly efficient and targeted swine fever nanovaccine, which can effectively improve the internalization, antigen presentation and immune response of the swine fever nanovaccine.

[0008] To solve the technical problem, the solution of the present invention is:

[0009] Provided is a highly efficient APC cell targeting polypeptide, referred to as PCT for short, which can specifically recognize and bind to the CD163 receptor of APC cells; the amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and its corresponding nucleotide sequence is shown in SEQ ID NO.2.

[0010] As a preferred embodiment of the present invention, the APC cells are porcine alveolar macrophages PAMs or Marc-145 cells, and the CD163 receptor is a specific receptor for APC cells.

[0011] The present invention also provides an APC cell-targeting nanoparticle mi3-PTC, which is formed by fusing the PCT polypeptide with the N-terminus of the nanoparticle mi3, expressing and purifying it in Escherichia coli and self-assembling it in vitro; the amino acid sequence of the nanoparticle mi3 is shown in SEQ ID NO.3, and its corresponding nucleotide sequence is shown in SEQ ID NO.4.

[0012] The present invention provides the use of the aforementioned polypeptide or nanoparticle mi3-PTC nanoparticle as a drug for promoting nanoparticle cellular internalization and antigen presentation.

[0013] The present invention also provides an APC cell-targeted classical swine fever virus self-assembly protein nanoparticle, which is obtained by fusion-linking and expressing the recombinant plasmid of the APC cell-targeted nanoparticle mi3-PTC with a classical swine fever virus antigen; the classical swine fever virus antigen is a B cell epitope, a T cell epitope, or a combination of the two CSFV EP on the classical swine fever E2 protein, and the respective amino acid sequences or corresponding nucleotide sequences are specifically shown in SEQ ID NOs. 5 to 12.

[0014] The present invention also provides a pharmaceutical composition comprising the aforementioned classical swine fever virus self-assembly protein nanoparticles and pharmaceutically acceptable excipients.

[0015] The present invention also provides the use of the aforementioned polypeptide, nanoparticle mi3-PTC, classical swine fever virus self-assembly protein nanoparticles or pharmaceutical composition in the preparation of a drug for preventing and / or treating classical swine fever virus infection, wherein the drug is a vaccine.

[0016] The present invention also provides a method for preparing a drug for preventing and / or treating classical swine fever virus infection, comprising: fusing and linking an APC cell-targeting polypeptide as shown in SEQ ID NO.1 with a nanoparticle carrier to express the APC cell-targeting nanoparticle mi3-PTC as shown in SEQ ID NO.3; and then further fusing and linking the APC cell-targeting nanoparticle with a classical swine fever virus antigen as shown in any one of SEQ ID NOs. 5 to 12 to express the APC cell-targeted classical swine fever virus self-assembling protein nanoparticle.

[0017] As a preferred embodiment of the present invention, the APC cell-targeted classical swine fever virus self-assembled protein nanoparticles are further mixed with pharmaceutically acceptable excipients to prepare a vaccine product for preventing and / or treating classical swine fever virus infection.

[0018] Compared with the prior art, the technical effects of the present invention are:

[0019] 1. The present invention displays an APC cell-targeting polypeptide on the surface of nanoparticle mi3, and carries the nanoparticles to APC cells expressing CD163, thereby significantly improving the internalization, storage, and distribution capabilities of the nanoparticles in vivo and in vitro.

[0020] 2. The swine fever virus self-assembled protein nanoparticles of the present invention can be used as a vaccine to significantly enhance the immune response to swine fever antigens.

[0021] 3. The swine fever virus self-assembly protein nanoparticles in the present invention can significantly improve the immune protection efficacy against swine fever virus, and can be used as a new swine fever virus particle vaccine to assist in the prevention and control of swine fever. Therefore, the present invention proposes a new development idea for targeted particle vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 To construct, express and characterize APC cell-targeted nanoparticles (mi3-PTC).

[0023] In the figure: A represents a schematic diagram of the screening of APC cell-targeting peptides derived from CD163 nanoantibody CDR3; B represents a schematic diagram of the construction of nanoparticles (mi3) and APC cell-targeting nanoparticles (mi3-PTC); C represents a diagram of the expression and purification results of mi3 and mi3-PTC; D represents an electron microscopy diagram of mi3 and mi3-PTC.

[0024] Figure 2 To evaluate the antigen internalization efficiency of mi3-PTC in vitro.

[0025] In the figure: A represents the result of mi3-PTC specifically binding to the CD163 receptor of APC cells; B represents the IFA result of mi3-PTC antigen internalization efficiency in Marc-145 cells; C represents the Western blot result of mi3-PTC antigen internalization efficiency in Marc-145 cells; D represents the grayscale analysis of the Western blot result of mi3-PTC antigen internalization efficiency in Marc-145 cells; E represents the IFA result of mi3-PTC antigen internalization efficiency in PAMs cells; F represents the Western blot result of mi3-PTC antigen internalization efficiency in PAMs cells; G represents the grayscale analysis of the Western blot result of mi3-PTC antigen internalization efficiency in PAMs cells.

[0026] Figure 3 To evaluate the antigen storage and distribution of mi3-PTC in vivo.

[0027] In the figure: A represents the time-dependent storage and distribution results of mi3-PTC antigen in mice; B represents the storage and distribution results of mi3-PTC in various organs of mice after 60 hours.

[0028] Figure 4 To construct, express and characterize APC cell-targeted swine fever nanovaccine preparation.

[0029] In the figure: A represents a schematic diagram of the construction of the APC cell-targeted classical swine fever nanovaccine (mi3-PTC-CSFV EP); B represents the expression and purification results of mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP proteins; C represents the electron microscopy results of mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP proteins.

[0030] Figure 5To evaluate the immunogenicity of APC cell-targeted classical swine fever nanovaccine in mice.

[0031] In the figure: A represents the ELISA test results of the level of classical swine fever-specific IgG in the mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP immunization groups; B represents the test results of the level of classical swine fever neutralizing antibodies in the mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP immunization groups; C represents the lymphocyte proliferation experiment results of the mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP immunization groups; D represents the experimental results of the number of specific IFN-γ secreting cells in spleen cells in the mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP immunization groups; E represents the qPCR results of the TNF-α cytokine level in the mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP immunization groups; F represents the qPCR results of the mi3-CSFV EP, GST-CSFV qPCR results of IL-4 cytokine levels in the EP and mi3-PTC-CSFV EP immunization groups; G represents the qPCR results of IL-10 cytokine levels in the mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP immunization groups.

[0032] Figure 6 To evaluate the immune protective efficacy of APC cell-targeted swine fever nanovaccine in rabbits.

[0033] In the figure: A represents the ELISA test results of the classical swine fever-specific IgG level in the GST-CSFV EP and mi3-PTC-CSFV EP immunization groups; B represents the test results of the classical swine fever neutralizing antibody level in the GST-CSFV EP and mi3-PTC-CSFV EP immunization groups; C represents the monitoring results of the body temperature change level of rabbits after the GST-CSFV EP and mi3-PTC-CSFV EP immunization groups were challenged with the virus; D represents the results of the classical swine fever virus load in the blood of the GST-CSFV EP and mi3-PTC-CSFV EP immunization groups 36h and 48h after the challenge; E represents the results of the classical swine fever virus load in the spleen after the GST-CSFV EP and mi3-PTC-CSFV EP immunization groups were challenged with the virus; F represents the immunohistochemistry results of the spleen after the GST-CSFV EP and mi3-PTC-CSFV EP immunization groups were challenged with the virus. DETAILED DESCRIPTION

[0034] This invention utilizes a nanobody phage library to screen for a nanobody CDR3 targeting peptide that targets the CD163 receptor on APCs. This targeting peptide is then surface-displayed on mi3 nanoparticles, effectively promoting mi3 nanoparticle internalization and antigen presentation. Furthermore, based on mi3-PTC, a method for preparing and applying a highly effective, targeted nanovaccine for swine fever is provided, overcoming the efficacy bottleneck of subunit vaccines.

[0035] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0036] Example 1 Construction, expression and characterization of APC cell-targeted nanoparticles (mi3-PTC)

[0037] (1) Construction of APC cell-targeted nanoparticles (mi3-PTC)

[0038] As attached Figure 1 As shown in the flowchart of Figure 1, after multiple rounds of affinity panning of the Nanobody phage library, Nanobodies that specifically recognize the CD163 receptor on APC cells are screened and the Nanobody sequences with the strongest specificity and binding are truncated. The APC cells are porcine alveolar macrophages (PAMs) or Marc-145 cells. Referring to existing literature, CDR3 is considered to be the fragment with binding ability of Nb, so the CDR3 functional domain is amplified as a targeting peptide sequence (abbreviated as PTC) targeting APC cells.

[0039] (2) Expression and purification of APC cell-targeted nanoparticles (mi3-PTC)

[0040] The PTC sequence was linked to the N segment of the self-assembling protein nanoparticle mi3, and the mi3-PTC segment was cloned into the PET30a vector. The ligation product was transformed into Rosetta (DE3) competent cells, and positive clones were selected and sequenced. The culture was expanded and cultured to OD 600nm=0.6, add IPTG to a final concentration of 1 mM, and induce overnight at 16°C. Collect the cells by centrifugation at 5000 rpm for 10 min at 4°C and resuspend in PBS buffer. Disrupt the cells using an ultrasonic cell disruptor in an ice bath, sonicating at 150 W for 60 min, 4 seconds on and 4 seconds off, until the cells are completely lysed. Subsequently, centrifuge at 12000 rpm for 15 min at 4°C, collect the supernatant, and remove impurities using a 0.22 μm cell strainer. Add the supernatant to a pre-equilibrated Ni-NTA agarose gel and allow to bind overnight on a shaker at 4°C. Add the protein-loaded agarose gel to a gravity-based protein purification column, and elute the target protein by affinity chromatography using a gradient of prepared imidazole dilution buffer (200 mM NaCl, 50 mM Tris, 50 mM-500 mM imidazole, pH 7.4). Collect the eluted target protein, aliquot, and store at -20°C.

[0041] (3) SDS-PAGE analysis: Prepare SDS-PAGE gel; take 60 μL of 500 mM imidazole eluate of mi3 and mi3-PTC proteins respectively, add 15 μL of 5× Loading Buffer, mix well by pipetting, and heat in a boiling water pot for 10 min; assemble the electrophoresis tank, fill it with electrophoresis solution, load the sample, and perform electrophoresis according to the procedure of 80V for 30 min and then 120V for 90 min; after the electrophoresis, place the gel in Coomassie brilliant blue dye solution, shake on a shaker at 37℃ for 2 h for staining, and then place it in decolorizing solution for decolorization, changing the decolorizing solution every 1 h. After the bands are clearly visible, place it on a gel imager to take pictures and observe the results.

[0042] (4) Western blot analysis: The 500mM imidazole eluate (containing the target protein) was subjected to SDS-PAGE electrophoresis. Transfer: Cut off a PVDF membrane of the same size as the polyacrylamide gel to be transferred, pre-treat the PVDF membrane with methanol for 2 minutes to activate its surface, and then transfer it to the wet transfer buffer for immersion. Assemble the transfer device in the order of (+) thick sponge-thin filter paper-PVDF membrane-gel-thin filter paper-thick sponge (-). Subsequently, the protein was transferred using the wet transfer method, and the constant current condition was set to 200mA for 50 minutes. After the transfer is completed, the PVDF membrane is placed in a blocking solution containing 5% skim milk powder TBST (Tris buffered saline solution, containing 0.1% Tween 20) and blocked at 37°C on a shaker for 1 hour. After the blocking is completed, the PVDF membrane is washed 3 times with TBST, each time for 5 minutes. Primary antibody incubation: Dilute mouse Flag monoclonal antibody 1:1000 in 0.5% skim milk powder and incubate overnight at 4°C on a shaker. Wash the PVDF membrane three times with TBST (5 min each). Secondary antibody incubation: Dilute HRP-conjugated goat anti-mouse secondary antibody 1:5000 in 0.5% skim milk powder and incubate at 37°C on a shaker for 1 h. Wash the PVDF membrane three times with TBST (5 min each). Add 200 μL of color development solution and photograph the gel using a gel imager.

[0043] (5) Transmission electron microscopy observation of mi3 and mi3-PTC nanoparticles: The purified target protein was transferred to a dialysis bag, and the imidazole solution environment was replaced with 10mM PBS at 4°C for 48 hours. The external PBS solution was replaced once every 24 hours. The dialyzed protein solution was added dropwise to the copper mesh and incubated for 30 seconds. The excess liquid was then absorbed with filter paper, and the copper mesh was allowed to air dry naturally. 2% phosphotungstic acid (PTA, Ph=6.8) was added dropwise to the copper mesh for negative staining for 60 seconds. The excess liquid was absorbed with filter paper, and the copper mesh was allowed to air dry naturally. The protein-loaded copper mesh was placed in a transmission electron microscope, and the self-assembly ability of the protein was observed at an accelerating voltage of 80kV and photographed for preservation.

[0044] Experimental results:

[0045] The CDR3 region of the nanobody that specifically recognizes the APC cell CD163 receptor (PTC fragment, 63 bp) was amplified and fused with the nanoparticle mi3 fragment and inserted into the PET30a vector (see attached). Figure 1-B), and verified by PCR and sequencing, recombinant PET30a-mi3 and PET30a-mi3-PTC plasmids were obtained. The recombinant plasmids were further introduced into the expression host strain Rosseta (DE3). After induction at 16°C, the mi3 and mi3-PTC target proteins were successfully purified by nickel column purification with high purity. The target proteins were approximately 25 kDa and 27 kDa, with protein concentrations of 0.18 mg / mL and 0.15 mg / mL, respectively. The SDS-PAGE and Western Blot results of the mi3 and mi3-PTC proteins are attached. Figure 1 -C. After further dialysis, the purified mi3 and mi3-PTC proteins were examined for their nanoparticle assembly using transmission electron microscopy. Transmission electron microscopy revealed that both mi3 and mi3-PTC proteins were able to self-assemble into nanoparticles (see Appendix Figure 1 -D).

[0046] Example 2 Evaluation of the in vitro antigen internalization efficiency of APC cell-targeted nanoparticles (mi3-PTC)

[0047] (1) APC cell targeting peptide can specifically bind to CD163 receptor

[0048] 3D4 cells, 3D4 CD163 When cells reach 90% of their 96-well plate, discard the old medium, wash twice with Hanks' solution, add 50 μL of 4% paraformaldehyde to each well, and fix at room temperature for 30 minutes. Discard the fixative, gently wash twice with PBS, add 50 μL of 0.2% Triton X-100 to each well, and permeabilize for 15 minutes at room temperature. Discard the permeabilization solution, gently wash twice with PBS, add 200 μL of 5% nonfat dry milk to each well, and block at 37°C for 2 hours. Discard the blocking solution, add purified mi3 and mi3-PTC to each well, and incubate at 37°C for 1 hour. Discard the supernatant, gently wash twice with PBS, and add a 1:1000 dilution of mouse Flag monoclonal antibody as the primary antibody to each well, and incubate at 37°C for 1 hour. Discard the supernatant, gently wash twice with PBS, add 50 μL of 1:2000 diluted FITC-labeled goat anti-mouse fluorescent secondary antibody to each well, and incubate at 37°C in the dark for 1 hour. Add 50 μL of 1:200 diluted DAPI to each well, stain the nuclei at room temperature for 5 minutes, gently wash twice with PBS, and observe and photograph under a fluorescence microscope.

[0049] (2) APC cell-targeting peptide promotes nanoparticle internalization

[0050] Marc-145 and PAMs cells with good growth status were selected and plated in 24-well culture plates (1×10 5Cells were incubated at 37°C for 3 hours, washed twice with Hanks' solution, and incubated in DMEM containing 2% serum for another 12 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with Triton-100 for 15 minutes, and blocked with goat serum for 15 minutes. Mouse Flag antibody was used as the primary antibody at a dilution of 1:1000 and incubated overnight at 4°C. FITC-conjugated goat anti-mouse fluorescent secondary antibody was then added at a dilution of 1:2000 for 1 hour at 37°C. Finally, nuclear staining with DAPI was performed at a dilution of 1:200 for 5 minutes. Immunofluorescence signals were detected using an inverted fluorescence microscope and a confocal laser scanning microscope (CLSM) IX81-FV1000 (Olympus), respectively.

[0051] Marc-145 and PAMs cells with good growth status were selected and plated in 24-well culture plates (1×10 5 Cells were added to the wells at a concentration of 40 μg / ml for mi3 and mi3-PTC nanoparticle proteins. The cells were incubated at 37°C for 3 hours, washed twice with Hanks' solution, and cultured in DMEM supplemented with 2% serum for another 12 hours. The supernatant was aspirated, and the cells were lysed with RIPA lysis buffer. The supernatant was collected by centrifugation at 4000 rpm. The protein concentration of the samples was determined using a BCA kit (Lianke Bio) and quantified. The quantified samples were added to 5× loading buffer and boiled for 10 minutes. The samples were separated by 12% SDS-PAGE. A 1:1000 dilution of mouse Flag monoclonal antibody was added to the transferred PVDF membrane and incubated overnight at 4°C on a shaker. The PVDF membrane was washed three times with TBST for 5 minutes each time. A 1:5000 dilution of HRP-conjugated goat anti-mouse secondary antibody was added and incubated at 37°C on a shaker for 1 hour. Western blots were visualized using SuperSignal West Pico Chemiluminescent Substrate, and images were captured on a Gel 3100 Chemiluminescent Imaging System, and the grayscale density of protein bands was analyzed using Gel-Pro Analyzer software.

[0052] Experimental results:

[0053] As attached Figure 2 -A shows that APC cell targeting peptide can specifically bind to CD163 receptor. Indirect immunofluorescence results showed that 3D4 cells, 3D4 CD163 In Marc-145 cells, compared with the control protein mi3, mi3-PTC showed obvious fluorescence, indicating that mi3-PTC can specifically bind to the CD163 receptor. In Marc-145 cells, compared with the control mi3, confocal microscopy results showed that the mi3-PTC internalization experimental group showed obvious fluorescent spots (see Appendix Figure 2-B), Western blot results showed that the internalization amount of mi3-PTC nanoparticles was significantly increased compared with the control group mi3 (see Appendix Figure 2 -C, D). In PAMs cells, compared with the control group mi3, confocal microscopy results showed that the mi3-PTC internalization experimental group had obvious fluorescent spots (see Appendix Figure 2 -E), Western blot results showed that the internalization amount of mi3-PTC nanoparticles was significantly increased compared with the control group mi3 (see Appendix Figure 2 -F, G). These results indicate that APC cell-targeting peptide (PTC) can significantly enhance the internalization of nanoparticles.

[0054] Example 3 Evaluation of mi3-PTC antigen storage and distribution levels in vivo

[0055] (1) Cy5 fluorescent labeling of mi3 and mi3-PTC proteins

[0056] Take the protein to be labeled (10 mg), dissolve it in 1 mL of pure water, add 0.1 mL of Coupling Reagent, mix well and set aside. Take 1 mg of Cy5-mix solid, dissolve it in 0.2 mL of Cy5-mix solution, add it to the protein solution and mix thoroughly. Avoid reaction at room temperature for 30 minutes (stirring or not), transfer the reaction solution to an ultrafiltration tube, and ultrafilter at 10,000 g for 5 minutes to remove unreacted Cy5-mix. To improve the purity, add pure water to suspend the protein and repeat the ultrafiltration 1-2 times. Resuspend the antibody / protein retained in the ultrafiltration tube with pure water or PBS to obtain a Cy5 fluorescently labeled antibody / protein solution. The fluorescence intensity of each sample was tested in vitro to ensure that the initial fluorescence intensity of each group was similar.

[0057] (2) Evaluate the differences in antigen storage and distribution of mi3 and mi3-PTC in mice

[0058] Six female BALB / c mice aged 6-8 weeks were randomly divided into two groups of three mice each. Mice were fed a diet without alfalfa for two weeks before immunization to reduce background autofluorescence. Each group of mice was injected subcutaneously in the back with 50 μg of mi3 and mi3-PTC, respectively. For in vivo imaging, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg / kg), and the in vivo biodistribution of the fluorescent antigen in each group was observed at different time points using an IVIS Lumina III Small Animal In vivo Imaging System (PerkinElmer, Waltham, MA). Mice were autopsied 60 h after injection, and the biodistribution of the fluorescent antigen in the heart, lungs, kidneys, spleen, and liver of each group of mice was studied.

[0059] Experimental results:

[0060] To investigate the in vivo retention and distribution of APC-targeted nanoparticles, the transport kinetics of mi3 and mi3-PTC proteins were investigated by in vivo imaging. First, Cy5 fluorescently labeled mi3 and mi3-PTC proteins were injected into the backs of BALB / c mice to assess local responses at the injection site. Compared with mi3, mi3-PTC induced a more persistent antigenic reservoir at the injection site (see Appendix). Figure 3 -A). The fluorescence signal of mi3-PTC was still detectable 60 hours after injection. 60 hours after injection, organs (heart, lung, kidney, spleen and liver) of mi3 and mi3-PTC immune groups were isolated for imaging. Figure 3 -B results showed that the fluorescence signal intensity in the heart, lungs, kidneys, spleen, and liver of the mi3-PTC-immunized group was significantly increased compared to the mi3-immunized group. These results indicate that the antigen expressed by mi3-PTC is stored in the body longer than the mi3 antigen and is more conducive to antigen delivery to secondary immune organs.

[0061] Example 4 Construction, expression and characterization of APC cell-targeted swine fever nanovaccine preparations

[0062] (1) Expression and purification of APC cell-targeted classical swine fever virus self-assembly protein nanoparticles (mi3-PTC-CSFV EP)

[0063] Based on literature research, two conserved B cell-neutralizing and T cell-neutralizing epitopes on classical swine fever virus were obtained. Classical swine fever epitope peptides (CSFV EPs) were synthesized by flexible ligation. The CSFV EPs were inserted into the PET30a-GST, PET30a-mi3, and PET30a-mi3-PTC plasmids. The ligation products were transformed into Rosetta (DE3) competent cells and expressed in prokaryotes. The target protein was purified using Ni-NTA, and a sample was analyzed by SDS-PAGE and Western blot. The target protein was collected, aliquoted, and stored at -20°C.

[0064] (2) Transmission electron microscopy observation of mi3-CSFV EP, GST-CSFV EP and mi3-PTC-CSFV EP nanoparticles: The purified target protein was transferred to a dialysis bag, and the imidazole solution environment was replaced with 10mM PBS at 4°C for 48 hours. The external PBS solution was replaced once every 24 hours. The dialyzed protein solution was added dropwise to the copper mesh and incubated for 30 seconds. The excess liquid was then absorbed with filter paper and the copper mesh was allowed to air dry naturally. 2% phosphotungstic acid (PTA, Ph = 6.8) was added dropwise to the copper mesh for negative staining for 60 seconds. The excess liquid was absorbed with filter paper and the copper mesh was allowed to air dry naturally. The protein-loaded copper mesh was placed in a transmission electron microscope, and the self-assembly ability of the protein was observed at an accelerating voltage of 80kV and photographed for preservation.

[0065] Experimental results:

[0066] A commercial company was commissioned to synthesize the classical swine fever epitope peptide (CSFV EP), which was then inserted into the PET30a-GST, PET30a-mi3, and PET30a-mi3-PTC plasmids. After verification by PCR and sequencing, the recombinant PET30a-mi3-CSFV EP, PET30a-GST-CSFV EP, and PET30a-mi3-PTC-CSFV EP plasmids were obtained (see attached). Figure 4 -A). The recombinant plasmid was further introduced into the expression host strain Rosseta (DE3). After induction at 16°C, the mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP target proteins were successfully obtained with high purity via nickel column purification. The target proteins were approximately 31 kDa, 34 kDa, and 34.4 kDa, with protein concentrations of 0.3 mg / mL, 0.26 mg / mL, and 0.22 mg / mL, respectively. The SDS-PAGE and Western Blot results of the mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP proteins are attached. Figure 4 -B. The purified mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP proteins were further dialyzed and then the nanoparticle assembly was detected using transmission electron microscopy. Transmission electron microscopy revealed that mi3-CSFV EP, GST-CSFV EP, and mi3-PTC-CSFV EP proteins were able to self-assemble into nanoparticles (see Appendix Figure 4 -C).

[0067] Example 5 Evaluation of the immunogenicity of APC cell-targeted swine fever nanovaccine in mice

[0068] (1) Animal immunization

[0069] Twenty six-week-old female BALB / c mice were randomly divided into four groups of five. Group 1 received a subcutaneous injection of 15 μg of mi3-CSFV EP protein, group 2 received a subcutaneous injection of 15 μg of GST-CSFV EP protein, group 3 received a subcutaneous injection of 15 μg of mi3-PTC-CSFV EP protein, and group 4 received a subcutaneous injection of PBS. A second immunization was performed 14 days after the first immunization, and blood samples were collected on days 0, 7, 14, 21, and 28 after the first immunization. Mice were sacrificed 35 days after immunization for spleen cell isolation.

[0070] (2) Indirect ELISA to detect the level of swine fever-specific antibodies

[0071] Mouse sera were collected on days 7, 14, 21, and 28 after immunization, and sera were assayed for classical swine fever-specific antibody levels by ELISA. The specific steps were as follows: CSFV EP was diluted to 5 μg / mL using coating solution, added to an ELISA plate (100 μL / well), and coated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with PBST (PBS, 0.05% Tween-20) buffer with shaking for 5 minutes each. Subsequently, 1% casein solution was added and incubated at 37°C for 2 hours. After washing three times with PBST buffer with shaking, sera from each immunization group were added at a 1:1000 dilution and incubated at 37°C for 1 hour. After washing three times with PBST, HRP-conjugated goat anti-mouse antibody was added at a 1:10,000 dilution and incubated at 37°C for 1 hour. After washing three times with PBST, TMB colorimetric solution (200 μL / well) was added and incubated at room temperature in the dark for 10 minutes. Subsequently, 2M H2SO4 (50 μL / well) was added, and the absorbance at 450 nm was read using a microplate reader.

[0072] (3) Detection of neutralizing antibodies against swine fever - fixed virus - diluted serum method

[0073] The day before, PK-15 cells were plated in a 96-well plate and cultured in a 37°C incubator. The cell density reached 80% on the second day. The serum samples to be tested were inactivated at 56°C for 30 minutes and serially diluted in DMEM medium in a 96-well cell culture plate. The initial dilution ratio was 1:2. 200 TCID 50 Mix equal volumes of the HZ08 strain / 100μL with the diluted serum sample and incubate in a 37°C incubator for 1 hour. Discard the culture medium in the 96-well plate, wash the cells three times with PBS, and add the above mixture to each well. Set up four parallel wells for each gradient. Also set up culture medium, serum, and virus controls, and incubate in a 37°C incubator for 1 hour. Discard the cell culture medium, wash the cells three times with PBS, and add DMEM containing 2% FBS to each well for maintenance. After 48 hours, evaluate the neutralizing effect of the serum by IFA. Calculate the titer of neutralizing antibodies in the serum to be tested by the Reed-Muench method.

[0074] (4) Lymphocyte proliferation test

[0075] On day 35 after immunization, spleen cells from mice in each group were isolated, and lymphocyte proliferation was assessed using a CCK-8 assay. The specific procedures were as follows: Splenic lymphocytes were seeded at a density of 105 cells / mL in a 96-well cell culture plate. CSFV EP antigen was added to each well of the immunization group as a stimulator, while concanavalin A (10 μg / mL) and RPMI 1640 medium were used as positive and negative controls. After 48 hours of static culture, 10 μL of CCK-8 reagent was added to each well, mixed, and incubated at 37°C for 4 hours. The absorbance at 450 nm was measured using a microplate reader, and the stimulation index (SI) was calculated according to the formula: SI = (OD450 value of the immunization group - OD450 value of the blank control) / (OD450 value of the negative control group - OD450 value of the blank control).

[0076] (4) Specific IFN-γ enzyme-linked immunosorbent spot (ELISpot) detection

[0077] On the 35th day after immunization, spleen cells from each group of mice were isolated and the level of nsp6-specific IFN-γ secreting cells was detected according to the instructions of the IFN-γ ELISpot kit. The specific steps are as follows: Mouse spleen cells were isolated and resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% antibiotics, and added to the ELISpot wells (10 5 cells / mL). CSFV EP protein was added to each well of spleen cells in the immunization group as an antigen stimulus, and concanavalin A (10 μg / mL) and RPMI1640 medium were set up as positive and negative controls. After static culture for 48 hours, the cell culture medium was discarded, the well plate was washed 5 times with PBS, and then 100 μL of r4-6a2-biotin antibody (1 μg / mL) diluted in PBS (containing 0.5% fetal bovine serum) was added to each well and incubated at room temperature for 2 hours. Subsequently, after washing the well plate 5 times with PBS, 100 μL of HRP-labeled streptavidin (1:1000) diluted in PBS (containing 0.5% fetal bovine serum) was added to each well and incubated at room temperature for 1 hour. After washing the well plate 5 times with PBS, 100 μL of TMB substrate solution was added to each well, and the cells were developed until obvious spots appeared. The spots were observed and counted using an EliSpot automatic counter.

[0078] (5) Cytokine level detection:

[0079] On day 35 after immunization, blood was collected from mice in each immunization group, and RNA was extracted from the blood using an RNA extraction kit. The RNA was reverse-transcribed into cDNA, and the relative mRNA levels of TNF-α, IL-4, and IL-10 were analyzed using qRT-PCR.

[0080] Experimental results:

[0081] As a potential nanovaccine targeting swine fever, the efficacy of mi3-PTC-CSFV EP was first evaluated in mice. Figure 5 As shown in Figure 2A, the indirect ELISA results showed that the 15μg mi3-CSFV EP-vaccinated group produced higher levels of CSFV-specific antibodies at 21dpi and 28dpi compared with the 15μg GST-CSFV EP-vaccinated group. Notably, the antibody level in the mi3-PTC-CSFV EP-vaccinated group was significantly higher than that in the mi3-CSFV EP and GST-CSFV EP groups. No CSFV-specific antibodies were detected in the PBS-vaccinated group. Figure 5 As shown in Figure 1-B, the results of the neutralization test showed that the 15μg mi3-CSFV EP vaccination group produced neutralizing antibodies against classical swine fever virus (HZ08) at 14dpi compared with the 15μg GST-CSFV EP vaccination group. It is worth noting that the mi3-PTC-CSFV EP vaccination group also produced neutralizing antibodies against classical swine fever virus (HZ08) at 14dpi, but the neutralizing antibody level was significantly higher than that of the mi3-CSFV EP vaccination group. 35 days after immunization, the results of the blood cytokine level test of mice showed that compared with the GST-CSFV EP vaccination group, the TNF-α, IL-4 and IL-10 levels of the mi3-PTC-CSFV EP vaccination group were significantly higher than those of the mi3-CSFV EP vaccination group and the GST-CSFV EP vaccination group (see Appendix). Figure 5 -E, F, G). To further explore the effect of APC cytokine peptide (PTC) on the proliferation of specific lymphocytes in response to classical swine fever antigens, spleen cells from mice in each immunization group were isolated on day 35 after inoculation, and the lymphocyte proliferation level of each immunization group was analyzed using a lymphocyte proliferation assay. The results showed that compared with the GST-CSFV EP vaccination group, the mi3-CSFV EP and mi3-PTC-CSFV EP groups induced significant lymphocyte proliferation. As shown in the attached figure, Figure 5 As shown in Figure 3-C, the SI index induced by the mi3-CSFV EP group was approximately 1.2 times that of the GST-CSFV EP group, while the SI index induced by the mi3-PTC-CSFV EP group was approximately 1.5 times that of the mi3-CSFV EP group. The IFN-γ ELISpot kit was used to detect the development of spleen cell-specific IFN-γ secreting cells in each immunization group. The results showed that compared with the GST-CSFV EP vaccination group, the mi3-CSFV EP and mi3-PTC-CSFV EP groups induced significant development of swine fever-specific IFN-γ secreting cells (see Appendix). Figure 5-D). These results indicate that APC cell peptide (PTC) can significantly enhance the immunogenicity of the swine fever nanovaccine.

[0082] Example 6 Evaluation of the immune protection efficacy of APC cell-targeted classical swine fever nanovaccine in rabbits.

[0083] (1) Animal immunization

[0084] Twelve New Zealand white rabbits weighing approximately 2 kg were randomly divided into four groups, with three rabbits in each group. Group 1 was subcutaneously immunized with 50 μg mi3-PTC-CSFV EP, Group 2 was subcutaneously immunized with 50 μg GST-CSFV EP, Group 3 was subcutaneously immunized with DMEM, and Group 4 was subcutaneously immunized with DMEM. A secondary immunization was performed 14 days after the first immunization, and blood samples were collected on days 0, 7, 14, 21, and 28 after the first immunization. 35 days after immunization, 10 μg of the immunized rabbits were injected intravenously into the ear margin of the first, second, and third immunization groups. 4 The TCID50 dose of strain C was administered to group 4, and the same dose of DMEM was injected into the ear vein as a negative control. The rectal temperature of the rabbits was monitored every 6 hours from 2 days before immunization until the 5th day after immunization.

[0085] (2) Determination of viral genome copy number by fluorescence quantitative PCR

[0086] The rabbits were killed on the 5th day after immunization, blood was collected and spleen was separated to extract total RNA, and the genome copy number of classical swine fever virus was detected by RT-PCR.

[0087] The total RNA extraction method was carried out according to the Novozymes total RNA extraction kit, and the specific steps are as follows: weigh a certain amount of spleen and homogenize it for 10 minutes, then centrifuge it briefly and carefully separate the supernatant; take 250 μL of the homogenate and add 750 μL of Trizol lysis buffer to lyse the cells at room temperature for 5 minutes; add 200 μL of chloroform solution, vortex mix for 5 minutes, and centrifuge at 12000 rpm at 4°C for 10 minutes; after aspirating the supernatant, add 1 / 2 volume of anhydrous ethanol and vortex mix, transfer it to a new RNA adsorption column, and centrifuge at 12000 rpm at 4°C for 1 minute; discard the waste liquid, add 600 μL of washing solution, and centrifuge at 12000 rpm at 4°C for 1 minute; repeat the previous step; discard the waste liquid, spin at 12000 rpm at 4°C for 2 minutes; add 40 μL of RNA-free H2O to the RNA-free EP tube, and centrifuge at 12000 rpm at 4°C for 2 minutes to collect the extracted RNA.

[0088] cDNA Synthesis: After total RNA samples were measured for concentration using a Nanodrop 2000 spectrophotometer, the RNA amount of each sample was adjusted to the same value using RNA-free H2O, based on the lowest sample concentration. Reverse transcription was performed using the Novozymes HiScript IIQ RT SuperMix (+gDNA wiper) kit. The primers are shown in Table 1. The downstream primers were used for both reverse transcription and quantitative fluorescence PCR, specifically for reverse transcription of the 5'-UTR gene of classical swine fever virus and β-actin mRNA in total RNA, respectively, to generate the corresponding cDNA samples.

[0089] Fluorescence quantitative PCR: A pair of specific primers were designed for the 5'-UTR of the CSFV genome and the internal reference gene β-actin, respectively. The amplified products were 129 bp and 114 bp in length, respectively. The reaction conditions were: initial denaturation at 95°C for 5 min; followed by 40 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 30 s. The pcDNA3-5'-UTR recombinant plasmid was used as the standard, and a 10-fold dilution was used to establish a standard curve. The detection range was 10 2 -10 9 copies / mL, added to the above system and reacted simultaneously.

[0090] Table 1 Primers used in qPCR

[0091]

[0092] (3) Immunohistochemistry

[0093] The spleen tissues of rabbits isolated on the 5th day after the rabbits were immunized with the virus were fixed with 4% paraformaldehyde for 24 hours and then subjected to immunohistochemistry. Immunohistochemistry was performed as follows: fixed animal tissues were paraffin-embedded and cut into approximately 5 μm thick paraffin sections. Paraffin sections were treated in xylene I for 10 minutes, followed by xylene II for 10 minutes. Sections were then treated in a gradient of ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol, with each step treated for 10 minutes. Sections were rinsed three times with pure water, the surface of the sections carefully wiped dry, and antigen retrieval was performed using microwave technology. Sections were rinsed three times with PBS and blocked with goat serum at 37°C for 2 hours to block nonspecific reactivity between the antigen and the antibody within the tissue. A monoclonal antibody against Classical Swine Fever E2 (1:100 dilution) was added, with PBS as a negative control, and the sections were incubated overnight at 4°C. An HRP-conjugated goat anti-swine secondary antibody (1:1000 dilution) was added and incubated at 37°C for 2 hours. The SABC complex was added and incubated at 37°C for 10 minutes. DAB staining was used for development. All experimental steps were performed in a humidified chamber. After immunostaining, the slides were counterstained with hematoxylin, differentiated with hydrochloric acid and alcohol, dehydrated with alcohol gradient, and transparentized with xylene, and finally sealed with neutral resin.

[0094] Experimental results:

[0095] After immunization, rabbits were blooded every 7 days through the anterior vena cava and serum was separated to detect changes in the level of swine fever-specific antibodies. Indirect ELISA results showed that the level of swine fever-specific antibodies gradually increased during the entire immunization cycle compared with the DMEM control group. It is worth noting that after 14 days of immunization, the antibody level in the mi3-PTC-CSFV EP vaccination group was significantly higher than that in the GST-CSFV EP vaccination group (see Appendix). Figure 6 -A). Neutralizing antibody test results showed that the neutralizing antibody level against classical swine fever virus in the mi3-PTC-CSFV EP vaccination group was significantly higher than that in the GST-CSFV EP vaccination group 14 days after immunization (see Appendix Figure 6 -B). 28 days after immunization, the neutralizing antibody level of mi3-PTC-CSFV EP vaccination group was as high as 1:256. 28 days after vaccination, rabbits were vaccinated through the ear vein for 10 4 TCID50 / rabbit C strain. 18-24h after the challenge, the rabbits in the GST-CSFV EP vaccination group and the C strain vaccination control group showed typical fever symptoms, which lasted for 18-20h, while the mi3-PTC-CSFV EP vaccination group did not show any typical symptoms during the entire experiment. On the 4th day after the challenge, the body temperature of all the challenge groups dropped to normal (see Appendix Figure 6 -C). Blood samples were collected from rabbits 36h and 48h after challenge to detect viremia. The results showed that the viral RNA loads of the challenge positive control group and the GST-CSFVEP vaccination group were 5.5×10 4 copies / μL, 3.0×10 4 The viral copy number of the mi3-PTC-CSFVEP vaccination group was only 42 copies / μL (see Appendix Figure 6 -D). Four days after the challenge, the spleen of rabbits was collected to detect the viral load of classical swine fever virus. The results showed that the viral RNA loads of the challenge positive control group and the GST-CSFV EP vaccination group were 5.8×10 4 copies / μL, 3.2×10 4 The viral copy number of the mi3-PTC-CSFV EP vaccination group was only 20 copies / μL (see Appendix Figure 6 -F). Further immunohistochemistry was used to detect whether CSFV virus particles were present in the spleen tissue of rabbits. Figure 6As shown in Figure 3-G, in addition to the positive signals of classical swine fever in the tissues of the challenge control group and the GST-CSFV EP vaccination group, no positive signals of classical swine fever were found in the mi3-PTC-CSFV EP vaccination group and the negative control group. These results indicate that APC cell-targeting peptides can significantly enhance the immune protection of classical swine fever nanovaccines.

[0096] In practical applications, the APC-targeted classical swine fever virus self-assembling protein nanoparticles are mixed with pharmaceutically acceptable excipients to produce the final vaccine product. For example, the final vaccine form can be an injection or spray; compatible excipients include Seppic 206 adjuvant, aluminum salt adjuvant, or Seppic 201 adjuvant. The concentration of the self-assembling protein nanoparticles in the vaccine is 0.2 mg / ml, and the recommended dosage is 0.05 mg / dose.

[0097]

[0098]

[0099]

Claims

1. A highly efficient APC cell targeting polypeptide, characterized in that: The polypeptide is referred to as PCT for short, and can specifically recognize and bind to the CD163 receptor of APC cells; the amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and its corresponding nucleotide sequence is shown in SEQ ID NO.

2.

2. The polypeptide according to claim 1, characterized in that The APC cells are porcine alveolar macrophages PAMs or Marc-145 cells, and the CD163 receptor is a specific receptor of the APC cells.

3. An APC cell-targeting nanoparticle mi3-PTC, characterized in that: The PCT polypeptide is fused with the N-terminus of the nanoparticle mi3, expressed and purified by E. coli, and self-assembled in vitro; the amino acid sequence of the nanoparticle mi3 is shown in SEQ ID NO.3, and its corresponding nucleotide sequence is shown in SEQ ID NO.

4.

4. Use of the polypeptide according to any one of claims 1 to 2 or the mi3-PTC nanoparticle according to claim 3 as a drug for promoting nanoparticle cellular internalization and antigen presentation.

5. An APC cell-targeted classical swine fever virus self-assembly protein nanoparticle, characterized in that: It is obtained by fusion expression of the recombinant plasmid of APC cell-targeted nanoparticle mi3-PTC and the classical swine fever virus antigen; the classical swine fever virus antigen is a B cell epitope, a T cell epitope or a combination of the two CSFV EP on the classical swine fever E2 protein, and the respective amino acid sequences or corresponding nucleotide sequences are specifically shown in SEQ ID NO.5 to 12.

6. A pharmaceutical composition, characterized in that It comprises the swine fever virus self-assembly protein nanoparticles as described in claim 5, and pharmaceutically acceptable excipients.

7. Use of the polypeptide according to any one of claims 1 to 2, the nanoparticle mi3-PTC according to claim 3, the classical swine fever virus self-assembly protein nanoparticles according to claim 5, or the pharmaceutical composition according to claim 6 in the preparation of a medicament for preventing and / or treating classical swine fever virus infection, wherein the medicament is a vaccine.

8. A method for preparing a drug for preventing and / or treating classical swine fever virus infection, characterized in that: include: The APC cell-targeting polypeptide shown in SEQ ID NO.1 is fused and linked to a nanoparticle vector for expression to obtain the APC cell-targeting nanoparticle mi3-PTC as a vector shown in SEQ ID NO.3; then it is further fused and linked to an APC cell-targeting APC cell-targeting APC cell-targeting APC cell-targeting nanoparticle mi3-PTC as shown in any one of SEQ ID NO.5 to 12 to obtain APC cell-targeted APC cell-targeted APC cell-targeted nanoparticle mi3-PTC.

9. The method according to claim 8, characterized in that The APC cell-targeted classical swine fever virus self-assembled protein nanoparticles are further mixed with pharmaceutically acceptable excipients to prepare a vaccine product for preventing and / or treating classical swine fever virus infection.

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