A nanoparticle using ARC protein as a carrier, its preparation method and application

Abstract

The present invention provides a nanoparticle using the ARC protein as a carrier, its preparation method and application. The nanoparticle is a nanoparticle formed by coupling a first recombinant protein containing the ARC protein with a second recombinant protein containing the polypeptide to be loaded and / or delivered and self-assembling. The present invention also provides the application of the nanoparticle in preparing vaccines and intracellular therapeutic drugs. The nanoparticle provided by the present invention delivers model antigens such as the RBD of the SARS-CoV-2 spike protein and the M1R of the monkeypox virus, can activate the production of highly efficient humoral and cellular immune responses, and has immune protection against lethal challenge in a mouse model. It is expected to be a safe, general and efficient endogenous recombinant protein delivery carrier to enhance the immunogenicity of target antigens.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology. Specifically, it relates to nanoparticles with an immune-enhancing effect, using ARC protein as a carrier, and their preparation methods and applications. Background Art

[0002] Activity-regulated cytoskeleton-associated protein (ARC) is a synaptic plasticity regulatory protein (NCBI NP_056008) derived from the Ty3 / gypsy retrotransposon family and is widely expressed in species such as humans, rats, and Drosophila. ARC consists of a positively charged N-terminal domain and a negatively charged C-terminal domain, which are homologous to the matrix and capsid domains of retroviral gag proteins. ARC can self-oligomerize and assemble into a virus-like capsid structure, participate in intercellular RNA transmission, and has the potential to be used as a novel vaccine carrier.

[0003] Nanocarriers play a prominent role in the design of novel vaccines, enabling multivalent display on the antigen surface, enhancing antigen-presenting cell uptake, and activating the generation of a highly efficient immune response. As an important vaccine carrier, virus-like particles (VLPs) have high biocompatibility and strong immunogenicity and have been widely used in the research and development of novel vaccines against pathogens such as hepatitis B virus and human papillomavirus. However, virus-derived self-assembling proteins are more likely to produce an immune imprinting effect, and the protective immune response may be inhibited to a certain extent when delivering other antigens as a molecular scaffold. Developing a self-assembling protein vaccine delivery carrier with low immunogenicity can further improve safety and immune specificity. The purpose of the present invention is to design and prepare a carrier based on ARC protein nanoparticles, which can efficiently activate the generation of a specific immune response against the target antigen through self-assembly and modular splicing strategies. Summary of the Invention

[0004] Based on the above purpose, the present invention first provides a nanoparticle using ARC protein as a carrier to load and / or deliver a polypeptide. The nanoparticle is a nanoparticle formed by coupling a first recombinant protein containing ARC protein with a second recombinant protein containing the loaded and / or delivered polypeptide and self-assembling. Among them, ARC protein is fused with a first molecular linker to form a first recombinant protein, and the polypeptide to be loaded and / or delivered is fused with a second molecular linker to form a second recombinant protein. The first molecular linker and the second molecular linker are a pair of molecular linking peptides that can specifically couple.

[0005] In the technical solution of the present invention, the ARC protein (activity-regulated cytoskeleton-associated protein), that is, the cytoskeletal activity regulatory protein, is a synaptic plasticity regulatory protein derived from the Ty3 / gypsy retrotransposon family, and the polypeptide refers to a compound formed by connecting more than 3 α -amino acids together by peptide bonds. The coupling between the proteins refers to the process in which two or more proteins are combined to form a complex through non-covalent chemical bonds (such as proline phosphorylation, protein ubiquitination, etc.) or covalent chemical bonds (such as disulfide bonds) by modifying the amino acid residues at specific sites of the proteins. The molecular linker peptide refers to two molecular connectors that can undergo the above-mentioned modifications to form non-covalent chemical bonds or covalent chemical bonds and couple with each other, that is, the first molecular connector and the second molecular connector.

[0006] In a preferred technical solution, the polypeptide includes one or more of antigens, antibodies, enzymes, and functional peptides. In the technical solution of the present invention, the functional peptide refers to a peptide substance that plays an important regulatory function in the body. Based on the concept of the present invention, the ARC protein can form nanoparticles through coupling with the above-mentioned antigens, antibodies, enzymes, and functional peptides and self-assembly.

[0007] In another preferred technical solution, the pair of molecular linker peptides is SpyTag peptide and SpyCatcher peptide. In the technical solution of the present invention, the SpyTag peptide and SpyCatcher peptide refer to polypeptide fragments that can spontaneously form isopeptide bonds isolated from the pilin protein of the Gram-positive bacterium Streptococcus pyogenes ( Streptococcus pyogenes ), which are respectively called SpyTag (16 amino acids) and SpyCatcher (113 amino acids), and the asp117 of SpyTag and the lys31 of SpyCatcher can spontaneously dehydrate to form an isopeptide bond. Those skilled in the art can understand that based on the concept of the present invention, using other paired molecular linker peptides in the art to fuse with the ARC protein and the polypeptide to be loaded and / or delivered respectively can also implement the present invention, that is, form nanoparticles self-assembled by the ARC protein and the polypeptide to be loaded and / or delivered.

[0008] In a more preferred technical solution, the ARC protein is fused with the SpyCatcher peptide to form a first recombinant protein, and the polypeptide is fused with the SpyTag peptide to form a second recombinant protein.

[0009] More preferably, the polypeptide is a novel coronavirus RBD antigen, a monkeypox virus M1R antigen or a monkeypox virus A35R antigen. The above three antigens are all specific embodiments of the present invention. Those skilled in the art can understand that based on the concept of the present invention, other immunogens for stimulating the body's immune system to produce protective antibodies can also be used for the antigens of the present invention to self-assemble with the ARC protein to form nanoparticles.

[0010] Particularly preferably, in the first recombinant protein, the ARC protein with the sequence shown in SEQ ID NO:1 is located at the C-terminus, and the SpyCatcher3 with the sequence shown in SEQ ID NO:7 is located at the N-terminus. The first recombinant protein is named "SC3-ARC protein"; and

[0011] In the second recombinant protein, the novel coronavirus RBD antigen with the sequence shown in SEQ ID NO:16 is located at the C-terminus, and the SpyTag3 with the sequence shown in SEQ ID NO:13 is located at the N-terminus. The second recombinant protein is named "RBD-ST3"; or

[0012] In the second recombinant protein, the monkeypox virus M1R antigen with the sequence shown in SEQ ID NO:19 is located at the N-terminus, and the SpyTag3 with the sequence shown in SEQ ID NO:13 is located at the C-terminus. The second recombinant protein is named "M1R-ST3"; or

[0013] In the second recombinant protein, the monkeypox virus A35R antigen with the sequence shown in SEQ ID NO:22 is located at the N-terminus, and the SpyTag3 with the sequence shown in SEQ ID NO:13 is located at the C-terminus. The second recombinant protein is named "A35R-ST3".

[0014] In another preferred technical solution, in the first recombinant protein, the ARC protein with the sequence shown in SEQ ID NO:1 is located at the C-terminus, and the SpyCatcher3 with the sequence shown in SEQ ID NO:7 is located at the N-terminus (SC3-ARC protein); and,

[0015] In the second recombinant protein, the cell-penetrating peptide TAT with the sequence shown in SEQ ID NO:10 is located at the C-terminus, and the SpyTag3 with the sequence shown in SEQ ID NO:13 is located at the N-terminus. The second recombinant protein is named "ST3-TAT".

[0016] Secondly, the present invention provides the use of the above-mentioned nanoparticles in the preparation of vaccines. The nanoparticles provided by the present invention are based on the self-assembly of ARC protein. The assembled and loaded antigens show enhanced immunogenicity in vivo. Moreover, both in vitro and in vivo presentation can activate antigen-presenting cells, and can enhance the residence time of antigens in vivo, prolong antigen accumulation and promote the maturation of DCs in vivo, showing its self-adjuvant effect. Therefore, the nanoparticles provided by the present invention show application prospects in the preparation of vaccines.

[0017] Thirdly, the present invention provides the use of the above-mentioned nanoparticles in the preparation of intracellular therapeutic drugs, and the nanoparticles are further loaded with drugs that need to be delivered into cells. One embodiment of the present invention shows that nanoparticles self-assembled based on ARC protein and the cell-penetrating peptide TAT can efficiently penetrate into HeLa cells, and the cell-penetrating effect is better than gene fusion. Therefore, based on the concept of the present invention, using nanoparticles conjugated and spliced with ARC protein and the cell-penetrating peptide TAT as carriers, the nanoparticles and the drugs to be delivered into cells can be used to deliver the drugs into the interior of target cells in a conjugated, gene fusion or labeled manner. Therefore, the above-mentioned nanoparticles provided by the present invention have application prospects in the preparation of intracellular therapeutic drugs.

[0018] Finally, the present invention provides a method for the above-mentioned nanoparticles, and the method includes the following steps:

[0019] (1) Prepare the first recombinant protein and the second recombinant protein respectively;

[0020] (2) Mix the first recombinant protein and the second recombinant protein.

[0021] In a preferred technical solution, in step (1), the first recombinant protein (SC3-ARC protein) is prepared with the coding sequence shown in SEQ ID NO: 9, and

[0022] the second recombinant protein (RBD-ST3) is prepared with the coding sequence shown in SEQ ID NO: 18, or

[0023] the second recombinant protein (M1R-ST3) is prepared with the coding sequence shown in SEQ ID NO: 21, or

[0024] the second recombinant protein (A35R-ST3) is prepared with the coding sequence shown in SEQ ID NO: 24, or

[0025] the second recombinant protein (ST3-TAT) is chemically synthesized with the sequence shown in SEQ ID NO: 15.

[0026] In the specific embodiments of the present invention, the above coding sequences are all optimized DNA sequences, and purification tags are added. Through the above method, high-purity recombinant proteins can be obtained, meeting the technical requirements for further preparation of nanoparticles.

[0027] In another preferred technical solution, in step (2), the molar ratio of the first recombinant protein to the second recombinant protein in the mixture is 0.5 - 2:1. In the specific embodiments of the present invention, depending on the different proteins to be loaded, the first recombinant protein and the second recombinant protein have different preferred mixing molar ratios. For example, SC3-ARC and RBD-ST3 are mixed at a molar ratio of 1:2, SC3-ARC and M1R-ST3 are mixed at a molar ratio of 1:1, SC3-ARC and A35R-ST3 are mixed at a molar ratio of 1:1, and SC3-ARC and ST3-TAT are mixed at a molar ratio of 2:1.

[0028] The present invention provides a vaccine delivery vector based on the ARC protein. Through modular splicing, it can self-assemble with protein antigens in vitro to form nanoparticles capable of efficiently multivalent display of antigens. Among them, the affinity of the antigen RBD loaded and displayed by the nanoparticles for its receptor is increased by more than 10 times. In the ARC-based nanoscaffold in the nanoparticles, it can enhance the in vivo residence time of antigens and prolong antigen accumulation. In the application of the RBD antigen of the novel coronavirus, the M1R-ARC nanoparticles significantly increase the proportion of MHCI + CD80 + 、MHCII + CD80 + and CD80 + CD86 + positive cells, promoting the maturation of antigen-presenting cells dendritic cells in vivo. In specific immunization applications, when the RBD-ARC, M1R-ARC, and A35R-ARC nanoparticles are used as vaccines, they can all stimulate the body to produce higher antigen-specific antibodies and neutralizing antibodies than the antigen alone, and produce a more balanced Th1 / Th2 immune activation response.

[0029] In the application of the nanoparticles provided by the present invention as a vaccine preparation, the nanoparticles can significantly increase the titers of specific antibodies and neutralizing antibodies. Among them, compared with the single RBD, the RBD-ARC nanoparticles induce a higher level of RBD-specific IgG titer, which increases by 16.2 times on the 28th day. In addition, comparing the IgG1 and IgG2a antibody titer ratios, compared with the single RBD antigen that induces a Th2-biased immune response, the RBD-ARC nanoparticles induce a more balanced Th1 / Th2 response. After the second immunization, the RBD-ARC nanoparticles induce a high level of neutralizing antibodies against SARS-CoV-2, with a titer reaching 1230, which is 56 times that of the single RBD antigen.

[0030] In the application of the monkeypox virus M1R antigen, compared with the single M1R, the M1R-ARC nanoparticles induce a higher level of M1R-specific immune response, which increases by 58.7 times on the 28th day. In addition, when the immunization dose of M1R-ARC is reduced to one-tenth of M1R-ST3, M1R-ARC can still induce a higher titer of M1R-specific IgG antibodies, further indicating the high immunogenicity of M1R-ARC. By infecting BS-C-1 cells with ectromelia virus (ECTV) to detect neutralizing antibodies, the results show that the neutralizing antibodies activated by M1R-ARC on the 28th day are 3.8 times that of M1R. The multiplex cytokine assay of cellular immunity shows that M1R-ARC induces higher levels of GM-CSF, IFN-γ, IL-2, and TNF-α compared with M1R-ST3, indicating that M1R-ARC has a more balanced Th1 / Th2 immune activation response. The IFN-γ cellular immunity results detected by enzyme-linked immunospot (ELISpot) show that M1R-ARC induces approximately 150 spots per million spleen cells, which is significantly higher than that of M1R-ST3. In the lethal challenge model of mousepox, each mouse was challenged intraperitoneally with 200 PFU of mousepox virus. Compared with the PBS or M1R groups, all mice in these groups died within 7 days or 18 days respectively, and the M1R-ARC injection provided a 68% protection efficiency. Extromelia virus, ECTV) to detect neutralizing antibodies, the results show that the neutralizing antibodies activated by M1R-ARC on the 28th day are 3.8 times that of M1R. The multiplex cytokine assay of cellular immunity shows that M1R-ARC induces higher levels of GM-CSF, IFN-γ, IL-2, and TNF-α compared with M1R-ST3, indicating that M1R-ARC has a more balanced Th1 / Th2 immune activation response. The IFN-γ cellular immunity results detected by enzyme-linked immunospot (ELISpot) show that M1R-ARC induces approximately 150 spots per million spleen cells, which is significantly higher than that of M1R-ST3. In the lethal challenge model of mousepox, each mouse was challenged intraperitoneally with 200 PFU of mousepox virus. Compared with the PBS or M1R groups, all mice in these groups died within 7 days or 18 days respectively, and the M1R-ARC injection provided a 68% protection efficiency.

[0031] In the application of the monkeypox virus A35R antigen, compared with A35R, A35R-ARC induces a stronger A35R-specific immune response, which increases by 58.7 times on the 28th day and 8.7 times on the 42nd day.

[0032] The nanoparticles self-assembled based on the ARC protein and the cell-penetrating peptide TAT provided by the present invention can efficiently penetrate into HeLa cells, and the cell penetration effect is better than that of gene fusion, indicating that it can be used as a carrier for intracellular drug delivery.

[0033] Therefore, the nanoparticles provided by the present invention are expected to serve as a safe, general, and efficient endogenous recombinant protein delivery vector, enhancing the immunogenicity of target antigens, and having application prospects as a vaccine preparation and an intracellular delivery vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 . Schematic diagram of the immune enhancement effect of ARC nanoparticles;

[0035] Figure 2 . SDS-PAGE, Western Blot detection, and particle size analysis of ARC;

[0036] Figure 3 . SDS-PAGE detection and particle size analysis of SC3-ARC;

[0037] Figure 4 . SDS-PAGE detection of gene fusion ARC-TAT and conjugated splicing TAT-ARC;

[0038] Figure 5 . Cellular delivery detection of conjugated splicing TAT-ARC and gene fusion ARC-TAT;

[0039] Figure 6 . SDS-PAGE detection of SC3-ARC and conjugated splicing RBD-ARC of RBD-ST3;

[0040] Figure 7 . Affinity detection of RBD, RBD-ST3, and RBD-ARC with hACE2;

[0041] Figure 8 . Enhancement of RBD immunogenicity by RBD-ARC nanoparticles;

[0042] Figure 9 . SDS-PAGE detection and particle size analysis of SC3-ARC and conjugated splicing M1R-ARC of M1R-ST3;

[0043] Figure 10 . Enhancement of M1R humoral immune response by M1R-ARC nanoparticles;

[0044] Figure 11 . Enhancement of M1R cellular immune response by M1R-ARC nanoparticles;

[0045] Figure 12 . Enhancement of lethality challenge protection against ectromelia virus by M1R-ARC nanoparticles;

[0046] Figure 13 . Enhancement of A35R immunogenicity by A35R-ARC nanoparticles;

[0047] Figure 14 . ARC nanoparticles stimulate dendritic cell maturation in vitro;

[0048] Figure 15 . ARC nanoparticles stimulate the secretion of pro-inflammatory factors by dendritic cells in vitro;

[0049] Figure 16 . ARC nanoparticles activate antigen-presenting cell maturation in vivo;

[0050] Figure 17 . ARC nanoparticles prolong the in vivo residence time of antigens;

[0051] Figure 18 . Adjuvant-free ARC nanoparticles prolong the in vivo residence time of antigens;

[0052] Figure 19 . Adjuvant-free M1R-ARC nanoparticles enhance the M1R humoral immune response;

[0053] Figure 20 . Adjuvant-free M1R-ARC nanoparticles enhance the protection against lethal challenge with monkeypox virus;

[0054] Figure 21 . Adjuvant-free M1R-ARC nanoparticles enhance the M1R cellular immune response;

[0055] Figure 22 . Detection of blood biochemical indexes in mice immunized with M1R-ARC nanoparticles;

[0056] Figure 23 . Histopathological examination of liver and kidney tissues in mice immunized with M1R-ARC nanoparticles. Detailed implementation manners

[0057] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the protection scope defined by the claims of the present invention.

[0058] The inventive concept of the present invention is as Figure 1 shown. A carrier based on ARC protein nanoparticles is designed and prepared, that is, the new coronavirus antigen (RBD) or the monkeypox virus antigen (M1R or A35R) and the ARC protein are self-assembled and modularly spliced through protein conjugation to form nanoparticles capable of efficiently multivalent displaying antigens. In the said nanoparticles, the ARC-based nanoscaffold can enhance the in vivo residence time of antigens, prolong antigen accumulation, and significantly increase MHC I in antigen-presenting cells dendritic cells + CD80 +, MHCII + CD80 + and CD80 + CD86 + The proportion of positive cells promotes the maturation of antigen - presenting dendritic cells in vivo. In specific immunization applications, when the RBD - ARC, M1R - ARC, and A35R - ARC nanoparticles are used as vaccines, they can all stimulate the body to produce higher antigen - specific antibodies and neutralizing antibodies than the single antigen alone, and produce a more balanced Th1 / Th2 immune activation response.

[0059] Example 1. Preparation and property characterization of protein nanoparticles

[0060] (1) Recombinant preparation of full - length ARC protein

[0061] The amino acid sequence of the full - length human ARC protein is SEQ ID NO: 1. After fusing the GST tag (SEQ ID NO: 2) and His tag (SEQ ID NO: 3) at the N - terminus and C - terminus respectively, the amino acid sequence is SEQ ID NO: 4. The codon - optimized nucleotide sequence is SEQ ID NO: 5. It is ligated into the pET - 21a(+) vector through the NdeI and XhoI restriction sites, and transfected into BL21(DE3) Escherichia coli to construct a prokaryotic expression engineering strain. Cultured at 37 °C until the logarithmic growth phase (OD 600 = 0.8), induced expression at 16 °C for 20 hours, and the final concentration of IPTG was 1 μM. After collecting the bacteria, they were sonicated and affinity - purified using a HisTrap excel chromatography column. The GST tag was cleaved by HRV 3C protease and purified using a GSTarp 4B chromatography column. SDS - PAGE and WB experiments showed that the size of the recombinant purified protein band was consistent with the expected ( Figure 2 of A).

[0062] (2) Self - assembly of ARC recombinant protein into nanoparticles

[0063] Dynamic light scattering was used to detect the particle size distribution of the recombinant protein. The diameter of ARC was 22.8 nm, and the particle size of the control BSA protein was 5.3 nm. After treatment with 0.5% SDS, the particle size of ARC decreased to less than 10 nm, indicating that ARC can form protein nanoparticles through intermolecular interactions ( Figure 2 of B).

[0064] Example 2. Multivalent display of antigens by protein nanoparticles

[0065] (1) Preparation of recombinant SC3 - ARC protein

[0066] After the N-terminus and C-terminus of ARC (SEQ ID NO: 1) were respectively fused with the SpyTag / SpyCatcher system SpyCatcher3 (SC3) sequence (SEQ ID NO: 7) and the StrepII tag (SEQ ID NO: 6), it was called SC3-ARC, whose amino acid sequence was SEQ ID NO: 8, and the codon-optimized nucleotide sequence was SEQ ID NO: 9. It was ligated into the pET-21a(+) vector through the NdeI and XhoI restriction sites, and transfected into the BL21(DE3) Escherichia coli prokaryotic expression engineering bacteria. It was cultured at 37 °C until the logarithmic growth phase (OD 600 = 0.8), induced to express for 20 hours at 16 °C, and the final concentration of IPTG was 1 μM. After collecting the bacteria, they were ultrasonically disrupted, and affinity purification was carried out using a StrepTrap HP chromatography column. SDS-PAGE experiments showed that the molecular weight of the recombinant purified protein was as expected ( Figure 3 A). Dynamic light scattering detected that the diameter of the SC3-ARC protein was 24.1 nm, indicating that it could self-assemble to form protein nanoparticles ( Figure 3 B).

[0067] (2) Preparation of the fusion protein of recombinant ARC and cell-penetrating peptide

[0068] The cell-penetrating peptide TAT (SEQ ID NO: 10) and the StrepII tag (SEQ ID NO: 6) were respectively fused to the N-terminus and C-terminus of ARC (SEQ ID NO: 1), called ARC-TAT, whose amino acid sequence was SEQ ID NO: 11, and the codon-optimized nucleotide sequence was SEQ ID NO: 12. It was ligated into the pET-21a(+) vector through the NdeI and XhoI restriction sites, and transfected into the BL21(DE3) Escherichia coli prokaryotic expression engineering bacteria. It was cultured at 37 °C until the logarithmic growth phase (OD 600 = 0.8), induced to express for 20 hours at 16 °C, and the final concentration of IPTG was 1 μM. After collecting the bacteria, they were ultrasonically disrupted, and affinity purification was carried out using a StrepTrap HP chromatography column. SDS-PAGE showed that the molecular weight of the recombinant purified protein was as expected ( Figure 4 A).

[0069] (3) Coupling and splicing of ARC and cell-penetrating peptide

[0070] Fusing the SpyTag / SpyCatcher system SpyTag3 (ST3) sequence (SEQ ID NO:13) and the linker sequence (SEQ ID NO:14) to the N-terminus of the TAT cell-penetrating peptide (SEQ ID NO:10), named ST3-TAT, whose amino acid sequence is SEQ ID NO:15. Chemically synthesize the ST3-TAT polypeptide, mix ST3-TAT and SC3-ARC protein (SEQ ID NO:8) in a molar ratio of 1:2 in PBS buffer, place at room temperature for 10 minutes, and SDS-PAGE experiment shows that the molecular weight of the conjugated and spliced protein TAT-ARC meets the expectation ( Figure 4 as shown in B).

[0071] (4) Efficient cellular delivery of ARC by cell-penetrating peptide

[0072] Culture HeLa cells overnight in a 96-well plate, and transfect with FITC-labeled ARC-TAT protein (gene fusion) and TAT-ARC protein (conjugated and spliced) at a concentration of 100 ng / μL per well respectively. After 8 hours, perform cell imaging detection. The conjugated and spliced method achieves efficient cellular delivery, and is significantly better than the gene fusion strategy ( Figure 5 )

[0073] (5) Preparation of recombinant RBD-ST3 protein

[0074] Fuse the ST3 sequence (SEQ ID NO:13) and the his tag (SEQ ID NO: 3) to the N-terminus and C-terminus of the RBD domain (SEQ ID NO:16) of the SARS-CoV-2 S protein respectively, named RBD-ST3, whose amino acid sequence is SEQ ID NO:17, and the codon-optimized nucleotide sequence is SEQ ID NO:18. Clone it into the pcDNA3.4 plasmid by molecular cloning, and use the Expi293F eukaryotic expression system to prepare the recombinant protein, and perform affinity purification using the HisTrap excel chromatography column.

[0075] (6) Self-assembly and affinity detection of RBD-ST3 protein and SC3-ARC protein

[0076] Mix the RBD-ST3 protein and the SC3-ARC protein in different ratios, react overnight at 4°C, and SDS-PAGE shows that the two can self-assemble and bind, named RBD-ARC protein. When the molar ratio is RBD-ST3:SC3-ARC = 2:1 ( Figure 6), SC3-ARC had the optimal loading efficiency for RBD-ST3. To further verify that ARC could correctly display its antigenic epitope after binding to RBD as a carrier, the affinity of the conjugated and spliced RBD-ARC was detected by surface plasmon resonance method. Compared with RBD, the addition of the ST3 tag did not affect the binding of RBD-ST3 to the human angiotensin-converting enzyme 2 (hACE2) receptor. In addition, the receptor affinity constant of RBD-ARC reached 1.9 x 10 -9 M, significantly higher than 1.6 x 10 -8 M of RBD ( Figure 7 ), indicating that the ARC nanoparticles achieved multivalent surface display of RBD.

[0077] Example 3. Design and immunogenicity evaluation of protein nanoparticle vaccines

[0078] (1) Enhancement of the immunogenicity of the COVID-19 antigen

[0079] RBD-ST3 was conjugated and spliced with SC3-ARC (molar ratio 2:1) to prepare RBD-ARC ( Figure 8 A). On days 0 and 14, BALB / c mice were intramuscularly injected with PBS (sham), SC3-ARC, RBD-ST3, and RBD-ARC mixed with aluminum adjuvant. The RBD content in RBD-ST3 and RBD-ARC was 5 μg, PBS and SC3-ARC were used as control groups, and the aluminum adjuvant dose was 50 μg. Serum was collected on days 14, 28, and 42 after the first immunization and the RBD-specific IgG antibody titer was detected by ELISA. Compared with RBD-ST3, RBD-ARC induced a higher level of RBD-specific IgG titer, which increased by 16.2-fold on day 28 ( Figure 8 B). In addition, by comparing the IgG1 and IgG2a antibody titer ratios, compared with RBD-ST3 which induced a Th2-biased immune response, RBD-ARC induced a more balanced Th1 / Th2 response ( Figure 8 C). Pseudovirus neutralizing antibody detection showed that after the second immunization, RBD-ARC induced a high level of COVID-19 neutralizing antibody, with a titer of 1230, which was 56 times that of RBD-ST3 ( Figure 8 D).

[0080] (2) Enhancement of the immunogenicity of the monkeypox antigen

[0081] The C-terminus and N-terminus of the truncated protein of monkeypox virus M1R (SEQ ID NO: 19) are respectively fused with the ST3 sequence (SEQ ID NO: 13) of the SpyTag / SpyCatcher system and the StrepII tag (SEQ ID NO: 6), which is called M1R-ST3. Its amino acid sequence is SEQ ID NO: 20, and the codon-optimized nucleotide sequence is SEQ ID NO: 21. It is molecularly cloned into the pcDNA3.4 plasmid, and the recombinant protein is prepared using the Expi293F eukaryotic expression system and affinity purified using a StrepTrap HP chromatography column. M1R-ST3 protein binds to SC3-ARC protein in different ratios to form M1R-ARC, reacts overnight at 4°C, and its binding efficiency is verified by SDS-PAGE ( Figure 9 A of it), and it is found that when the molar ratio is M1R-ST3:SC3-ARC = 1:1, the optimal loading efficiency can be achieved. The particle size distribution is detected by dynamic light scattering, and the particle size results show that M1R-ARC can self-assemble to form protein nanoparticles ( Figure 9 B of it).

[0082] M1R-ARC is prepared by coupling and splicing M1R-ST3 with SC3-ARC (molar ratio is 1). At 0 day and 14 days respectively, BALB / c mice are intramuscularly injected with M1R-ST3, M1R-ARC, SC3-ARC and PBS mixed with aluminum adjuvant. The M1R content in M1R-ST3 and M1R-ARC is 5 μg, PBS and SC3-ARC are the control groups, and the aluminum adjuvant dose is 50 μg. Serum is collected on the 14th, 28th and 42nd days after the first immunization and the M1R-specific IgG antibody titer is detected by ELISA. Compared with M1R-ST3, M1R-ARC induces a higher level of M1R-specific immune response, which increases by 58.7 times on the 28th day ( Figure 10 A of it). In addition, when the immunization dose of M1R-ARC is reduced to one-tenth of M1R-ST3, M1R-ARC can also induce a higher M1R-specific IgG antibody titer ( Figure 10 A of it), further indicating the high immunogenicity of M1R-ARC. Neutralizing antibodies are detected by infecting BS-C-1 cells with ectromelia virus ( Extromelia ECTV), and the results show that the neutralizing antibodies activated by M1R-ARC on the 28th day are 3.8 times that of M1R-ST3 ( Figure 10 B of it). Multicytokine determination of cellular immunity shows that M1R-ARC induces higher levels of GM-CSF, IFN-γ, IL-2 and TNF-α compared with M1R-ST3, indicating that M1R-ARC has a more balanced Th1 / Th2 immune activation response ( Figure 11A). Enzyme-linked immunosorbent spot (ELISpot) assay for IFN-γ cellular immunity results showed that M1R-ARC induced approximately 150 spots per million splenocytes, significantly higher than the approximately 30 spots induced by M1R-ST3 ( Figure 11 B). In the lethal mousepox challenge model, each mouse was challenged intraperitoneally with 200 PFU of mousepox virus. Compared with the PBS or M1R-ST3 groups where all mice died within 7 days or 18 days respectively, the M1R-ARC injection provided 68% protection efficiency ( Figure 12 ).

[0083] The C-terminus and N-terminus of the monkeypox virus A35R protein (SEQ ID NO:22) were respectively fused with the SpyTag / SpyCatcher system ST3 sequence (SEQ ID NO:13) and the StrepII tag (SEQ ID NO: 6), named A35R-ST3. Its amino acid sequence is SEQ ID NO:23, and the codon-optimized nucleotide sequence is SEQ ID NO:24. It was molecularly cloned into the pcDNA3.4 plasmid, and the recombinant protein was prepared using the Expi293F eukaryotic expression system and affinity purified using a StrepTrap HP chromatography column. The A35R-ST3 protein and the SC3-ARC protein were combined in different ratios to form A35R-ARC, and the reaction was carried out overnight at 4°C. Its binding efficiency was verified by SDS-PAGE ( Figure 13 A). It was found that the optimal loading efficiency could be achieved at a molar ratio of A35R-ST3:SC3-ARC = 1:1. A35R-ARC was prepared by coupling and splicing A35R-ST3 and SC3-ARC (molar ratio of 1). On days 0 and 14, BALB / c mice were intramuscularly injected with A35R-ST3, A35R-ARC, SC3-ARC, and PBS mixed with aluminum adjuvant. The A35R content in A35R-ST3 and A35R-ARC was 5 μg, PBS and SC3-ARC were used as control groups, and the aluminum adjuvant dose was 50 μg. Serum was collected on days 14, 28, and 42 after the first immunization and the RBD-specific IgG antibody titer was detected by ELISA. Compared with A35R-ST3, A35R-ARC induced a stronger A35R-specific immune response, which increased by 58.7-fold on day 28 and 8.7-fold on day 42 ( Figure 13 B).

[0084] Example 4. Protein nanoparticles have self-adjuvant effects

[0085] (1) Activating antigen-presenting cells by in vitro delivery

[0086] Dendritic cells (DCs) play an important role in antigen uptake and presentation of vaccines. Bone marrow-derived dendritic cells (BMDCs) were extracted from BALB / c mice. M1R-ST3 and M1R-ARC were added to BMDCs at a concentration of 10 μg / mL and incubated for 20 hours. PBS and lipopolysaccharide (LPS) were used as negative and positive controls, respectively. Flow cytometry and cytokine detection showed that M1R-ARC could significantly activate BMDC maturation, highly express CD40, CD80, and CD86 ( Figure 14 A-C thereof) and secrete pro-inflammatory factors such as TNF-α, IL-6, and IL-1β. The concentrations of TNF-α, IL-6, and IL-1β detected in the cell supernatant were 2618.1 pg / mL, 4927.1 pg / mL, and 669.6 pg / mL, respectively, which were increased by 23.4-fold, 117.0-fold, and 48.0-fold compared with the M1R-ST3 treatment group ( Figure 15 A-C thereof).

[0087] (2) In vivo delivery to activate antigen-presenting cells

[0088] Activation of specific immune responses in vivo depends on the uptake of antigens by dendritic cells and their migration to lymph nodes. On days 0 and 14, BALB / c mice were intramuscularly injected with M1R-ST3, M1R-ARC, SC3-ARC, and PBS mixed with aluminum adjuvant. The M1R content in M1R-ST3 and M1R-ARC was 5 μg, and PBS and SC3-ARC were used as control groups. The dose of aluminum adjuvant was 50 μg. At 36 hours after the second immunization, the inguinal lymph nodes of mice were extracted, and the expression levels of co-stimulatory molecules and MHC molecules in the CD11c+ cell population were detected by flow cytometry. The proportions of MHCI+ CD80+, MHCII+ CD80+, and CD80+CD86+ positive cells in DC cells of the M1R-ARC treatment group were 3.4%, 2.5%, and 2.7%, respectively, which were increased by 2.6-fold, 3.5-fold, and 2.7-fold compared with the M1R-ST3 treatment group ( Figure 16 A-C thereof).

[0089] (3) In vivo delivery to prolong antigen retention time

[0090] Alexa Flura 750 fluorescently labeled M1R-ST3 and M1R-ARC. BALB / c mice were intramuscularly injected with fluorescently labeled M1R-ST3 or M1R-ARC mixed with aluminum adjuvant. The M1R content in M1R-ST3 and M1R-ARC was 5 μg, and the dose of aluminum adjuvant was 50 μg. At 48 h after administration, the inguinal lymph nodes were dissected, and the fluorescence signal of M1R-ARC was higher than that of M1R-ST3 ( Figure 17A). In vivo live imaging results showed that M1R-ST3 was rapidly cleared, with only 20.0% remaining at the injection site after 8 hours, while M1R-ARC showed higher fluorescence signals at different time points. At 8, 24, 48, and 72 hours, 78.2%, 35.4%, 16.8%, and 9.9% of the fluorescence signals remained at the injection site, respectively ( Figure 17 B and C), indicating that the ARC-based nanoscaffold can enhance the in vivo residence time of antigens, prolong antigen accumulation, and promote DC maturation in vivo.

[0091] Example 5. Immunogenicity evaluation of adjuvant-free protein nanoparticle vaccines

[0092] BALB / c mice were intramuscularly injected with fluorescently labeled M1R-ARC mixed with aluminum adjuvant (aluminum adjuvant dose was 50 μg) or adjuvant-free fluorescently labeled M1R-ARC, with the M1R-ARC dose being 5 μg in both cases. In vivo fluorescence imaging showed that adjuvant-free M1R-ARC could also remain in the body for a long time (12.3 hours vs 15.9 hours) ( Figure 18 A and B).

[0093] On days 0 and 14, BALB / c mice were intramuscularly injected with M1R or M1R-ARC mixed with aluminum adjuvant (aluminum adjuvant dose was 50 μg), or adjuvant-free M1R or M1R-ARC, with the M1R and M1R-ARC doses being 5 μg in both cases. Whether or not aluminum adjuvant was added, the antibody response induced by M1R-ARC was significantly higher than that of the M1R control group. On day 42, the M1R-specific IgG titers induced by adjuvant-free M1R-ARC and aluminum adjuvant-containing M1R-ARC were 1000 times and 31.6 times that of the M1R group, respectively. Moreover, compared with adjuvant-free M1R which could not induce a significant antibody response, adjuvant-free M1R-ARC had immunogenicity comparable to that of aluminum adjuvant-containing M1R-ARC. On day 42, the M1R-specific IgG titers induced by adjuvant-free M1R-ARC and aluminum adjuvant-containing M1R-ARC were 4.0 x 10 5 and 2.5 x 10 5 ( Figure 19 A). Detection of vaccinia virus (VACV) neutralizing antibodies showed that the level of neutralizing antibodies induced by adjuvant-free M1R-ARC was 7.4 times that of the M1R control group ( Figure 19 B). In the mousepox lethal challenge model, each mouse was intraperitoneally challenged with 200 PFU of mousepox virus. All mice in the M1R-ST3 control group died within 10 days, while adjuvant-free M1R-ARC could provide 100% immune protection ( Figure 20 ). In addition, by comparing the IgG1 and IgG2a antibody titer ratios, adjuvant-free M1R-ARC induced a more balanced Th1 / Th2 response ( Figure 21of A), and can induce a high level of T cell response. The IFN-γ cellular immune results detected by enzyme-linked immunosorbent spot (ELISpot) showed that M1R-ST3 induced approximately 12 spots per million splenocytes, and M1R-ARC induced approximately 82 spots ( Figure 21 of B).

[0094] Example 6. Safety evaluation of protein nanoparticle vaccine

[0095] On days 0 and 14 respectively, BALB / c mice were intramuscularly injected with M1R-ST3 or M1R-ARC mixed with aluminum adjuvant, where the M1R content in M1R-ST3 and M1R-ARC was 5 μg, and the aluminum adjuvant dose was 50 μg. On the 7th day after two-dose immunization, blood biochemical indexes and histopathology of the kidneys and liver were detected. Among the blood biochemical indexes of the control group and M1R-ARC-immunized mice, aspartate glutamate transaminase (ALT) was 43.4 U / L and 38.8 U / L respectively, aspartate aminotransferase (AST) was 133.4 U / L and 139.6 U / L respectively, alkaline phosphatase (ALP) was 146.6 U / L and 134.2 U / L respectively, blood urea nitrogen (urea) was 5.9 mmol / L and 6.2 mmol / L respectively, creatine (CR) was 14.6 μmol / L and 13.4 μmol / L respectively, total bilirubin (TBil) was 0.7 μmol / L and 0.7 μmol / L respectively, total globulin (TG) was 2.4 mmol / L and 1.8 mmol / L respectively, and total protein (TP) was 52.1 g / L and 53.8 g / L respectively. There was no significant difference in blood biochemical indexes between the two groups ( Figure 22 ), and no obvious histopathological changes were observed in the liver and kidneys ( Figure 23 ).

Claims

1. A nanoparticle that uses ARC protein as a carrier to load and / or deliver antigens, characterized in that: The nanoparticles are formed by coupling and self-assembling a first recombinant protein containing ARC protein and a second recombinant protein containing a loaded and / or delivered antigen, wherein the ARC protein is fused with a first molecular linker to form a first recombinant protein, and the loaded and / or delivered antigen is fused with a second molecular linker to form a second recombinant protein, the first molecular linker and the second molecular linker are a pair of molecular connecting peptides that can be specifically coupled, and the pair of molecular connecting peptides are SpyTag peptide and SpyCatcher peptide, the ARC protein is fused with SpyCatcher peptide to form a first recombinant protein, the antigen is fused with SpyTag peptide to form a second recombinant protein, and the antigen is a novel coronavirus RBD antigen, a monkeypox virus M1R antigen, or a monkeypox virus A35R antigen.

2. The nanoparticle according to claim 1, characterized in that In the first recombinant protein, the ARC protein with a sequence as shown in SEQ ID NO: 1 is located at the C-terminus, and the SpyCatcher3 with a sequence as shown in SEQ ID NO: 7 is located at the N-terminus; and, In the second recombinant protein, the novel coronavirus RBD antigen with a sequence such as SEQ ID NO: 16 is located at the C-terminus, and the SpyTag3 with a sequence such as SEQ ID NO: 13 is located at the N-terminus; or In the second recombinant protein, the monkeypox virus M1R antigen with a sequence as shown in SEQ ID NO: 19 is located at the N-terminus, and the SpyTag3 with a sequence as shown in SEQ ID NO: 13 is located at the C-terminus; or In the second recombinant protein, the monkeypox virus A35R antigen whose sequence is shown in SEQ ID NO:22 is located at the N-terminus, and the SpyTag3 whose sequence is shown in SEQ ID NO:13 is located at the C-terminus.

3. Use of the nanoparticles according to claim 1 or 2 in the preparation of vaccines.

4. A method for preparing the nanoparticles according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) preparing a first recombinant protein and a second recombinant protein respectively; (2) Mixing the first recombinant protein and the second recombinant protein.

5. The method according to claim 4, characterized in that In step (1), a first recombinant protein is prepared using the coding sequence shown in SEQ ID NO: 9, and The second recombinant protein is prepared using the coding sequence as shown in SEQ ID NO: 18, or The second recombinant protein is prepared using the coding sequence as shown in SEQ ID NO: 21, or The second recombinant protein is prepared using the coding sequence as shown in SEQ ID NO: 24, or The second recombinant protein was chemically synthesized according to the sequence shown in SEQ ID NO:

15.

6. The method according to claim 4, characterized in that In step (2), the molar ratio of the first recombinant protein to the second recombinant protein is 0.5-2:1.

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