Subunit vaccine based on DNA origami technology

Abstract

The invention discloses a subunit vaccine based on a DNA origami structure, a preparation method of the subunit vaccine, a pharmaceutical composition containing the subunit vaccine and application of the subunit vaccine to preparation of drugs for preventing or treating pathogen infection.

Description

Subunit vaccine based on DNA origami technology Technical Field

[0001] The present invention relates to the field of vaccines, and in particular to a subunit vaccine based on a DNA origami structure. Background Art

[0002] DNA origami consists of one or more template strands and numerous short strands that self-assemble through complementary base pairing [Saccà B, Niemeyer CM. DNA origami: the art of folding DNA. Angewandte Chemie (International ed in English). 2012;51:58-66]. Due to the high controllability of DNA strands, DNA origami is morphologically programmable and can be assembled into nanostructures of varying sizes and morphologies. Furthermore, due to the specificity of DNA sequences, DNA origami is surface addressable, and the short strands of DNA origami can be designed as capture strands through sequence extension and end-modification, enabling molecular connections with controllable distances, numbers, and patterns.

[0003] The widespread prevalence of pathogenic coronaviruses, represented by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has severely jeopardized human health. Several vaccines, employing different approaches, have been developed to combat the coronavirus disease (COVID-19) pandemic caused by SARS-CoV-2. Among them, subunit vaccines containing only key viral proteins have become the most numerous candidate SARS-CoV-2 vaccines in clinical and preclinical research due to their well-defined composition and high safety profile.

[0004] Although there are reports on RBD monomer vaccines [Yang J, et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature. 2020; 586: 572-79; Zhang NN, et al. A Thermostable mRNA Vaccine against COVID-19. Cell. 2020; 182: 1271-83. e16], the low molecular weight and surface low valence limit its immunogenicity.RBD multimerization is a popular and useful strategy to overcome these shortcomings, such as dimerization through Fc fragment fusion or disulfide bonds, and trimerization through stabilization of the T4 fibrin fold domain [Yang S, et al. Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: two randomized, double-blind, placebo-controlled, phase 1 and 2 trials. The Lancet Infectious diseases. 2021; 21: 1107-19; Gu H, et al. Adaptation of SARS-CoV-2 in BALB / c mice for testing vaccine efficacy. Science (New York, NY). 2020; 369: 1603-7; Liu Z, et al. RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response. Signal transduction and targeted 2020;5:282;Walsh EE,et al.Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates.The New England journal of medicine.2020;383:2439-50;Mulligan MJ,et al.Phase I / II study of COVID-19 RNA vaccine BNT162b1 in therapy adults.Nature.2020;586:589-93].

[0005] In recent years, in order to further increase the size and surface valence of antigens, methods of displaying multiple RBD copies on nanoparticles have attracted increasing attention. However, the size of these nanovaccines (10-30 nm) is significantly smaller than that of severe acute respiratory syndrome coronavirus 2 (~90 nm), and the evenly distributed surface antigen pattern differs from the clustered distribution of authentic severe acute respiratory syndrome coronavirus [Kanekiyo M, et al. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature. 2013; 499: 102-6; Boyoglu-Barnum S, Ellis D, Gillespie RA, Hutchinson GB, Park YJ, Moin SM, et al. Quadrivalent influenza nanoparticle vaccines induce broad protection. Nature. 2021; 592: 623-8; Tan TK, et al. A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralizing antibody responses. Nature communications. 2021; 12: 542; Ke Z, et al. Structures and distributions of SARS-CoV-2 spike proteins on intact virions. Nature. 2020; 588: 498-502]. Nanoparticles with controlled size, morphology, and surface patterns are urgently needed to improve the similarity between RBD nanovaccines and severe acute respiratory syndrome coronavirus SARS-CoV-2 and explore the structure-activity relationship between surface antigen patterns and vaccine efficacy.

[0006] Summary of the Invention

[0007] In one aspect, the present invention provides a subunit vaccine based on a DNA nanostructure, which comprises a DNA origami structure and antigenic peptides, wherein the antigenic peptides are clustered on the DNA origami structure, the number of antigenic peptides in each cluster is 2-6 and the distance between the antigenic peptides is 5-20 nm, and the number of clusters each DNA origami structure has is 1-20.

[0008] In one aspect, the present invention provides a pharmaceutical composition comprising the subunit vaccine of the present invention and a pharmaceutically acceptable carrier.

[0009] In one aspect, the present invention provides use of the subunit vaccine of the present invention in the preparation of a medicament for preventing or treating pathogen infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1: Design and construction of ICO-RBD nanovaccine. (a) Schematic diagram of the "engraving printing strategy" for modifying RBD antigens onto ICO. The genomic DNA of the M13mp18 phage was folded together with the staple and capture strands (with overhanging polyA) through an annealing process to form ICO as the nanocore. SpyTag-N3 and DBCO-polyT were covalently linked through a click chemistry reaction to form a SpyTag-polyT conjugate. The SpyTag peptide was then engraved on the ICO through complementary pairing between polyT and polyA. The fusion protein SpyCatcher-RBD was site-specifically linked to the ICO through the spontaneous formation of a covalent isopeptide bond between SpyCatcher and SpyTag. (b) The optimal surface antigen pattern was screened based on the efficiency of the nanovaccine in activating RBD-specific IgG-BCR (B-RBD cells) in vitro, and the effective short-term and long-term immune responses in vivo were evaluated. Various surface antigen patterns with different parameters were screened, including antigen spacing (10-40 nm), antigen copies within a cluster (1-5), and number of clusters (1-12). (cf) Characterization of ICO, including electrophoresis (c), TEM (d), AFM (e), and DLS (f) analysis. Scale bar (white), 1 μm. Scale bar (black), 100 nm. (gh) Gel electrophoresis (g) and antigen loading efficiency (n=4) (h) of ICO-RBD nanovaccines surface-modified with 12, 24, 36, 48, and 60 RBD antigens. Data were processed in GraphPad Prism 8 and presented as mean ± SD.

[0011] Figure 2: Effects of antigen spacing and cluster number on the efficiency of ICO-RBD nanovaccines in activating B-RBD cells. (a) Schematic diagram of the design of two series of ICO-RBD nanovaccines, including 10-40 nm antigen spacing and 1-12 antigen clusters. (b) Antigen binding to trigger BCR activation and BCR-dependent Ca2+ Signal transduction. Antigens, such as RBD, bind to the BCR on the surface of B cells, and intracellular Ca 2+ The increase in Ca concentration is caused by the activation of phospholipase Cγ2 (PLCγ2), which hydrolyzes phosphatidylinositol-4,5-bisphosphate to produce the second messenger inositol-1,4,5-triphosphate (IP3). When IP3 binds to and opens the IP3 receptor (IP3R) on the endoplasmic reticulum (ER), the Ca 2+ Stored Ca is depleted. Matrix interaction molecule 1 (STIM1) translocates to the plasma membrane, and then stored Ca 2+ The entry of Ca2+ (SOCE) is activated, and the BCR activates Ca 2+ Flow through the Orai1 channel. (c) Design and AFM images of ICO-RBD nanovaccine with 5 RBDs within 1 RBD cluster, but RBD spacing ranging from 10 to 40 nm. (d) Binding affinity between SpyCatcher-RBD protein (RBD) or ICO-RBD nanovaccine and B-RBD cells with different RBD spacing (n=3). ICO-RBD nanovaccine binding to B-RBD cells was detected using anti-His antibody and flow cytometry. (e) Ca in B-RBD cells triggered by SpyCatcher-RBD protein (RBD) or ICO-RBD nanovaccine with different RBD spacing, as detected by Fluo-4AM labeling and flow cytometry. 2+ (f) Design and AFM images of ICO-RBD nanovaccines with 1-12 RBD clusters, 5 RBD copies within the cluster, and RBD spacing of 10 nm. (g) Binding affinity between ICO-RBD nanovaccines with different numbers of RBD clusters and B-RBD cells (n=3). ICO-RBD nanovaccines bound to B-RBD cells were detected using anti-His antibodies and flow cytometry. (h) Ca2+ in B-RBD cells triggered by ICO-RBD nanovaccines with different numbers of RBD clusters, as detected by Fluo-4AM labeling and flow cytometry. 2+ Data were processed using GraphPad Prism 8 and presented as mean ± SD. Statistical significance (P value) was calculated using one-way ANOVA and Tukey's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, P > 0.05, indicating no statistically significant difference.

[0012] Figure 3: Humoral immune responses of BALB / c mice immunized with ICO-RBD nanovaccine. (a) Schematic diagram of the vaccination scheme and group information. Mice were randomly divided into 8 groups and primed (first shot) or boosted (second shot) with different vaccines at weeks 0 and 3. Red drops indicate blood sampling, and serum was collected every three weeks. All mice were euthanized at week 14 and memory immune cells were evaluated. (bc) SARS-CoV-2 RBD-specific IgG antibody titers in serum measured by ELISA at week 2 (b) and week 5 (c) (n=8). (d) Time-antibody titer curve of RBD-specific IgG titers measured every three weeks (n=8). (ef) Pseudovirus neutralization test of sera collected at weeks 5 and 14 to achieve an inhibitory dilution value (ID) for 50% neutralization 50 ) (n=6). (g) RBD in spleen cells of immunized mice at week 14 detected by flow cytometry + IgG + MBCs (B220 + CD38 + ) percentage (n=6). (hi) RBD at week 14 + Bone marrow plasma cells (BMPC, B220 - CD138 + ) and RBD + Splenic plasma cells (SPPC, B220 - CD138 + LLPCs (EpCAM hi CXCR3 - ) percentage (n=6). (j) Flow cytometry analysis of CD3 in spleen cells at week 14. + CD8 + Central memory T cells (CD44 + CD62L + ) percentage (n = 6). Data are shown as mean ± SEM in Figures b, f. Data are presented as mean ± SD for groups g, j. Data were processed using GraphPad Prism 8. Statistical significance (P value) was calculated by one-way ANOVA and Tukey's test. **, P < 0.01; ***, P < 0.001. ns, P > 0.05, indicating no statistically significant difference.

[0013] Figure 4: T cell responses induced by ICO-RBD nanovaccine in mice. Mice were randomly divided into 3 groups and primed / boosted with different vaccines at week 0 and week 3. The spleen and lungs of mice were harvested at week 3 (before booster immunization) and week 6. After obtaining single cells, the cells were stimulated with SpyCatcher-RBD protein overnight. (ab) CD3 in the spleen detected by intracellular cytokine staining and flow cytometry + CD8 + T cells (a) and CD3 + CD4 + IFN-γ in T cells (b) + / IL-2 + / TNF-α + The proportion of cells in lung tissue was detected by intracellular cytokine staining and flow cytometry. + CD8 + T cells (c) and CD3 + CD4 + IFN-γ in T cells (d) + / IL-2 + / TNF-α + (ef) Intracellular cytokine staining analysis and flow cytometry analysis of CD3 in spleen (e) and lung tissue (f). + CD4 + IL-4 in T cells + (g) Detection of CD8 in lung tissue by flow cytometry. + CD69 in T cells + CD103 + Percentage of tissue-resident memory T cells (TRM) (n = 6). Data were processed using GraphPad Prism 8 and presented as mean ± SD. Statistical significance (P value) was calculated by one-way ANOVA and Tukey's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, P > 0.05, indicating no statistically significant difference.

[0014] Figure 5: Antigen presentation and germinal center response in draining lymph nodes induced by ICO-RBD nanovaccine. (a) ICO-RBD nanovaccine accumulates in lymph nodes. BALB / c mice were intramuscularly immunized with Cy5.5-labeled RBD monomer (SpyCatcher-RBD protein) or Cy5.5-labeled ICO-RBD nanovaccine. Twelve hours later, inguinal lymph nodes were obtained and then subjected to fluorescence imaging. (b) CD11C in inguinal lymph nodes detected by flow cytometry. +Cy5.5 in DC + DC, CD4 + Cy5.5 + Resident DC, CD21 / CD35 + Cy5.5 + Follicular DC, and F4 / 80 + Cy5.5 in macrophages + Percentage of macrophages (n=6). (c) Schematic diagram of the experimental timeline for analyzing germinal center reactions. BALB / c mice were randomly divided into PBS, RBD, and ICO-RBD groups. Inguinal lymph nodes were collected at 1, 2, and 3 weeks after priming. (d) CD4 + T cells FH cells (CXCR5 + PD1 + ), CD19 + B220 + GC B cells (CD95 + GL7 + ), plasma cells (CD44 + CD138 + ) percentage (n=8). Data were processed on GraphPad Prism 8 and expressed as mean ± SD. Statistical significance (P value) was calculated by one-way ANOVA and Tukey test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0015] Figure 6: Gel electrophoresis of ICO. (a) Gel electrophoresis of ICO containing 12-60 capture strands with overhanging poly A. (b) Gel electrophoresis of ICO containing 12-60 SpyTag (ST) or SpyTag-SpyCatcher-RBD (ST-SC-RBD).

[0016] Figure 7: Schematic diagram of the pATX2-SpyCatcher-RBD construct (a), Coomassie blue staining (b), and Western blot analysis of the fusion protein (c). SP, signal peptide. SC, Spycatcher. Linker: SEQ ID NO: 236. NPE, soluble fraction. MW, molecular weight.

[0017] Figure 8: Gel electrophoresis and antigen coverage percentage of ICO-RBD nanovaccines with different surface antigen patterns. (ab) Electrophoretic characterization (a) and antigen loading efficiency (b) of ICO-RBD nanovaccines with different RBD spacing (n=3). (cd) Electrophoretic characterization (c) and antigen loading efficiency percentage (d) of ICO-RBD nanovaccines with 1-12 RBD clusters, 5 RBD copies within the cluster, and RBD spacing of 10nm (n=3). (ef) Electrophoretic characterization (e) and antigen loading efficiency percentage (f) of ICO-RBD-6c×n-10nm nanovaccines with 1-5 antigen copies within the cluster (n=3). Data were processed on GraphPad Prism 8 and expressed as mean ± SD.

[0018] Figure 9: Effect of cluster antigen copies on the ability of ICO-RBD nanovaccines to activate B-RBD cells. (a) Design of ICO-RBD nanovaccine with 6 RBD clusters, 1-5 RBD copies within a cluster, and 10 nm RBD spacing. (b) Ca in B-RBD cells triggered by SpyCatcher-RBD protein (RBD) or ICO-RBD nanovaccine with different antigen copies within the cluster. 2+ Traces. (c) Binding affinity between SpyCatcher-RBD protein (RBD) or ICO-RBD nanovaccine with different cluster antigen copies and B-RBD cells (n=3). SpyCatcher-RBD protein (RBD) or ICO-RBD nanovaccine binding to B-RBD cells was detected using anti-His antibody and flow cytometry. Data were processed on GraphPad Prism 8 and expressed as mean ± SD. Statistical significance (P value) was calculated by one-way ANOVA and Tukey test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, P > 0.05, the difference was not statistically significant.

[0019] Figure 10: Immune cell analysis at week 14 (see Figure 3). (ab) RBD-specific bone marrow cells (a) BMPCs (B220 - CD138 + ) and SPPCs (B220 in RBD-specific splenocytes (b). - CD138 + (c) CD3 in spleen cells at week 14 detected by flow cytometry + CD8 + Effector memory T cells (CD44 + CD62L -) percentage (n = 6). Data were processed in GraphPad Prism 8 and expressed as mean ± SD. Statistical significance (P value) was calculated by one-way ANOVA and Tukey's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, P > 0.05, the difference was not statistically significant.

[0020] Figure 11: Cy5.5 expression in different DCs in lymph nodes detected by flow cytometry 12 hours after immunization + The proportion of cells. (a) CD11C + CD80 in DCs + Cy5.5 + Mature DCs (n=6). (b) CD11C + CD86 in DCs + Cy5.5 + Mature DCs (n=6). (c) CD11C + CD8a in DCs + Cy5.5 + Resident DCs (n=6). (d) CD11C + CD103 in DC + Cy5.5 + Migrated DCs (n=6). (e) CD11C + CD103 in DCs - Cy5.5 + Resident DCs (n = 6). Data were processed using GraphPad Prism 8 and presented as mean ± SD. Statistical significance (P value) was calculated by one-way ANOVA and Tukey's test. **, P < 0.01; ***, P < 0.001. ns, P > 0.05, no statistically significant difference.

[0021] Figure 12: SARS-CoV-2 RBD-specific IgG titers in serum after priming vaccination. Mice were randomly divided into three groups, and serum was collected weekly. (a) RBD-specific IgG titers (n = 8). (b) Time-dependent antibody titer curves (n = 8). Data were processed in GraphPad Prism 8 and presented as mean ± SEM.

[0022] Figure 13: In vivo immunogenicity of the ICO-HA influenza A antigen peptide nanovaccine. (a) ICO-HA nanovaccine immunization schedule; phosphate-buffered saline (PBS), HA monomer, ICO, and HA+ICO (physical mixture) were used as controls; (b) HA-specific IgG serum titers detected by enzyme-linked immunosorbent assay (ELISA) at week 2 after priming; (c) ICO-HA nanovaccine-induced HA-specific IgG average antibody titers after boosting.

[0023] Figure 14: In vivo immunogenicity of the ICO-CSP malaria antigen peptide nanovaccine. (a) ICO-CSP nanovaccine immunization schedule; phosphate-buffered saline (PBS), CSP monomer, ICO, and CSP monomer + ICO (physical mixture) were used as controls; (b) CSP-specific IgG serum titers detected by enzyme-linked immunosorbent assay (ELISA) two weeks after priming; (c) CSP-specific IgG average antibody titers elicited by the ICO-CSP nanovaccine after boosting.

[0024] Figure 15: In vivo immunogenicity of the ICO-P72 African swine fever recombinant antigen peptide nanovaccine. (a) ICO-P72 nanovaccine immunization schedule; phosphate-buffered saline (PBS), P72 recombinant antigen protein, ICO, and P72 recombinant antigen protein + ICO (physical mixture) were used as controls, respectively. (b) P72-specific IgG serum titers detected by enzyme-linked immunosorbent assay (ELISA) at week 2 after priming. (c) P72-specific IgG titers elicited by the ICO-P72 nanovaccine after booster immunization.

[0025] Figure 16: In vivo immunogenicity of the ICO-gB cytomegalovirus antigen peptide nanovaccine. (a) ICO-gB nanovaccine immunization schedule; phosphate-buffered saline (PBS), gB monomer, ICO, and gB + ICO (physical mixture) were used as controls; (b) gB-specific IgG serum titers measured by enzyme-linked immunosorbent assay (ELISA) at week 2 after priming; (c) average gB-specific IgG titers elicited by the ICO-gB nanovaccine after booster immunization.

[0026] Figure 17: In vivo immunogenicity of the ICO-F antigen peptide (SEQ ID NO: 195) nanovaccine. (a) ICO-F nanovaccine immunization schedule; phosphate-buffered saline (PBS), F protein monomer, ICO, and F protein + ICO (physical mixture) were used as controls; (b) F protein-specific IgG serum titers detected by enzyme-linked immunosorbent assay (ELISA) two weeks after priming; (c) F protein-specific IgG titers elicited by the ICO-F nanovaccine after boosting.

[0027] Figure 18: In vivo immunogenicity of the ICO-gE (SEQ ID NO: 196) nanovaccine. (a) ICO-gE nanovaccine immunization schedule; phosphate-buffered saline (PBS), gE protein monomer, ICO, and gE protein + ICO (physical mixture) were used as controls; (b) gE protein-specific IgG serum titers detected by enzyme-linked immunosorbent assay (ELISA) two weeks after priming; (c) gE-specific IgG titers elicited by the ICO-gE nanovaccine after boosting.

[0028] Summary of the Invention

[0029] Unless otherwise noted, the scientific and technical terms used herein should have the meanings commonly known to those skilled in the art. In addition, unless otherwise required, singular terms should include plural terms, and plural terms should include singular terms. The aforementioned techniques and methods are generally carried out according to conventional methods well known in the art and described in the references cited in this specification. See, for example, Sambrook et al.Molecular Cloning:A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)) and Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J.Wiley & Sons (New York, NY 1994), which are incorporated by reference; Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, are hereby incorporated by reference in their entirety.

[0030] As used herein, the terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably to refer to a polymer of any length, for example, two or more amino acid residues joined by peptide bonds. The term also includes amino acid polymers modified naturally or by human intervention; for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a tag or a biologically active component. Conventional single-letter or three-letter amino acid residue codes are used herein.

[0031] Herein, when describing amino acid sequences, unless otherwise specified or clear from the context, the amino acid sequences are referred to in the N-terminal to C-terminal direction.

[0032] As used herein, the terms "nucleic acid," "DNA," "polynucleotide," and "nucleotide sequence" are used interchangeably and refer to deoxyribonucleic acid, a linear or circular polymer of deoxyribonucleotides (deoxyadenine (A), deoxyguanine (G), deoxycytosine (C), and deoxythymidine (T)) connected by 3', 5'-phosphodiester bonds. The DNA may be single-stranded or double-stranded, linear or circular. Furthermore, the DNA may contain suitable modifications known in the art, such as methylation, phosphorothioate, and interruptions by non-nucleotide components.

[0033] When describing nucleic acid sequences herein, reference to nucleic acid sequences is in the 5' to 3' direction unless otherwise specified or the context indicates otherwise. Generally, reference to DNA (e.g., scaffold DNA, short DNA strands, complementary DNA strands, capture DNA strands, etc.) herein refers to single-stranded DNA unless otherwise specified or the context indicates otherwise.

[0034] Sequence identity can be determined by commercially available computer programs that employ any suitable algorithm to calculate the percent identity between two or more sequences, for example, using default parameters. A typical example of such a computer program is CLUSTAL. More advantageously, the BLAST algorithm is employed with the parameters set to default values. A detailed description of the BLAST algorithm is available on the National Center for Biotechnology Information (NCBI) website.

[0035] In this study, we employed DNA origami as a platform for the rational design and precise assembly of RBD nanovaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We first constructed icosahedral DNA origami (ICO) with a diameter of approximately 90 nm, which approximates the morphology and size of SARS-CoV-2 (Figure 1a). We then designed various surface antigen patterns with different parameters, including antigen spacing, number of antigen copies within a cluster, and number of clusters, and based on these designs, precisely modified RBD antigens onto ICOs (ICO-RBD) using an “engraving-printing” strategy (Figure 1a). In vitro experiments using B cells stably expressing cognate IgG receptors for RBD antigens were performed to explore the effects of these parameters on B cell activation and to screen nanovaccines with optimal surface RBD patterns (Figure 1b). We then systematically evaluated the short- and long-term immune effects of the optimized ICO-RBD nanovaccines in a mouse model (Figure 1b).

[0036] In a first aspect, a subunit vaccine based on a DNA origami structure is provided, which comprises a DNA origami structure and antigenic peptides, wherein the antigenic peptides are clustered on the DNA origami structure, the number of antigenic peptides in each cluster is 2-6 and the distance between the antigenic peptides is 5-20 nm, and the number of clusters each DNA origami structure has is 1-20.

[0037] As used herein, a DNA origami structure refers to a structure in which a long single-stranded DNA (usually genomic DNA) is base-complemented with a series of designed short DNA (staple) segments to controllably construct a desired pattern or structure. For example, DNA nanostructures designed from an M13 template strand can be designed using the open-source caDNAno software and annealed using a general program. Small nanostructured DNA nanostructures can be directly designed and annealed based on sequence complementarity, as described, for example, in Paul WK Rothemund, Folding DNA to create nanoscale shapes and patterns, Nature V440:297-302 (16 March 2006); RP Goodman et al., Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication, Science, V310:1661-1664 (9 December 2005).

[0038] The DNA origami structure used herein can be any DNA origami structure, particularly a three-dimensional structure having a size and shape similar to the pathogen from which the antigenic peptide is derived, such as a polyhedron (preferably a convex polyhedron, such as a polyhedron having at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 faces), such as an icosahedron. As described herein, a polyhedral DNA origami structure refers to a three-dimensional structure in which double-stranded DNA forms the edges of the polyhedron.

[0039] In one embodiment, the DNA origami structure used in this article is a 4-20-hedron, such as a 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19- or 20-hedron, preferably a tetrahedron, a hexahedron, an octahedron, a dodecahedron or an icosahedron, particularly preferably an icosahedron, such as a regular icosahedron.

[0040] In one embodiment, the diameter of the DNA origami structure used herein is between about 20-100 nm, for example, between about X-Y nm, wherein X is an integer selected from 20-50 (e.g., 20, 25, 30, 35, 40, 45, or 50) and Y is an integer selected from 60-100 (e.g., 60, 65, 70, 75, 80, 85, 90, 95, or 100). In one embodiment, the diameter of the DNA origami structure used herein is between about 80-95 nm, for example, about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 nm.

[0041] As described herein, the diameter of a DNA origami structure theoretically refers to the diameter of the smallest sphere that can enclose the DNA origami structure. The smallest sphere that encloses the DNA origami structure refers to a sphere with the smallest diameter that can position the site of the DNA origami structure inside or on the surface of the sphere. In one embodiment, the diameter is the hydrophilic diameter of the DNA origami structure detected using dynamic light scattering (DLS).

[0042] In one embodiment, the edge length of the DNA origami structure used herein is between about 100-150 base pairs (bp), for example, between about AB (bp), wherein A is an integer selected from 100-120 (e.g., 100, 105, 110, 115, or 120), and B is an integer selected from 130-150 (e.g., 130, 135, 140, 145, or 150). In one embodiment, the edge length of the DNA origami structure used herein is between about 110-120 bp, for example, about 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 bp.

[0043] In a further embodiment, the DNA origami structure is a DNA origami structure formed by short-chain DNA (i.e., Staple chain) shown in SEQ ID NO: 1-193 with M13 genomic DNA (e.g., SEQ ID NO: 203) as a scaffold, preferably, one or more of the short-chain DNAs further have an overhanging polyA (e.g., SEQ ID NO: 234) at the 5' end or 3' end to connect to the antigenic peptide connected to ployT through AT complementarity. In a further embodiment, the DNA origami structure is a DNA origami structure (having an overhanging polyA portion) formed by the short chain DNA shown in SEQ ID NO: 1, 3-14, 16-28, 30-41, 43-51, 53-54, 56-57, 59-64, 66-70, 72-81, 83-87, 89-93, 96-101, 103-107, 109-113, 116-121, 123-127, 129-130, 132-142, 144-152, 154-158, 160-164, 167-180, 182, 184-185, 187-190, 193 and the DNA shown in SEQ ID NO: 204-233 (with a capture chain with an overhanging polyA).

[0044] As used herein, the clustered distribution of antigenic peptides on the DNA origami structure refers to the relative aggregation of the sites where several antigenic peptides are connected to the DNA origami structure, wherein the minimum distance between each of the several antigenic peptides in a cluster and the remaining peptides in the cluster is less than or equal to a threshold value. If the minimum distance between an antigenic peptide and the antigenic peptides of a cluster is greater than the threshold value, then the antigenic peptide is not an antigenic peptide of the cluster. If the minimum distance between an antigenic peptide and the antigenic peptides of a cluster is less than or equal to the threshold value, then the antigenic peptide is an antigenic peptide of the cluster. In particular, the antigenic peptides are outwardly oriented on the DNA origami structure.

[0045] In one embodiment, the threshold value is about 5-20nm, for example about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20nm. In particular, if the antigen peptides are evenly distributed in the cluster (i.e., the distance minimums of each antigen peptide and all the other peptides are equal or approximately equal), the threshold value can be equal to the distance minimum. The threshold value should be less than the distance between two clusters.

[0046] In this article, mentioning the distance between two clusters refers to the minimum value of the distance between the antigenic peptide of one cluster and the antigenic peptide of another cluster, i.e. the distance between the two closest antigenic peptides in the two clusters respectively. In one embodiment, the distance between the two clusters is greater than the threshold value, for example, 1.5, 2, 2.5, 3, 3.5 times or more of the threshold value.

[0047] As used herein, the distance between two antigenic peptides refers to the straight-line distance between the two sites at which the two antigenic peptides are attached to the DNA origami structure. The site at which the antigenic peptide is attached to the DNA origami structure refers to the position of a nucleotide residue on the edge of the double-stranded DNA forming the polyhedral DNA origami structure, at which the residue is directly or indirectly (e.g., via a molecular linker) attached to the antigenic peptide (e.g., covalently or non-covalently). In particular, in some embodiments, the residues on the edge of the polyhedral DNA origami structure can be extended by connecting to a first single-stranded DNA, forming a double strand with a second single-stranded DNA that is at least partially, preferably completely, complementary to the DNA origami structure through the Watson-Crick base pairing principle (i.e., A / T, G / C pairing) (excluding the nucleotides forming the polyhedral edge), thereby connecting the DNA origami structure to the molecule (e.g., antigenic peptide) attached to the second single-stranded DNA; in this case, the site at which the antigenic peptide is attached to the DNA origami structure refers to the residue on the edge of the polyhedral DNA origami structure that is directly (e.g., covalently or non-covalently) attached to the first single-stranded DNA.

[0048] In one embodiment, the antigenic peptides of a cluster are evenly distributed, i.e., the distance between any one antigenic peptide in the cluster and the nearest other antigenic peptide is equal or approximately equal, for example, the sites where the antigenic peptides are attached to the DNA origami structure form vertices of a regular polygon such as an equilateral triangle, a square, or a regular pentagon. In one embodiment, the distance between the antigenic peptides in the cluster is about 5-20, 5-15, 5-10 nm, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably about 5, 6, 7, 8, 9, or 10 nm, more preferably about 10 nm.

[0049] In one embodiment, the number of antigenic peptides in each cluster is 2-6, for example 2, 3, 4, 5 or 6 antigenic peptides. In one embodiment, each site where the antigenic peptides in a cluster are connected to the DNA origami structure is evenly distributed on the perimeter of a regular polygon, for example, a site is about 5-20, 5-15, 5-10 nm away from another site nearest to the cluster, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably about 5, 6, 7, 8, 9 or 10 nm, more preferably about 10 nm. In one embodiment, the site where the antigenic peptides in a cluster are connected to the DNA origami structure forms the vertices of a regular polygon such as an equilateral triangle, a regular quadrilateral, a regular pentagon or a regular hexagon, and more preferably, the length of the side of the regular polygon is about 10 nm. The antigenic peptides may be antigenic peptides from the same pathogen, which may have the same or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identical amino acid sequences. Preferably, the antigenic peptides have the same amino acid sequences.

[0050] In one embodiment, the number of antigenic peptides in a cluster is 5, and preferably, the sites where these 5 antigenic peptides are connected to the DNA origami structure form 5 vertices of a regular pentagon, and more preferably, the side length of the regular pentagon is about 10 nm.

[0051] As used herein, each DNA origami structure has 1-20 clusters, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, each DNA origami structure has at least 6 antigenic peptide clusters, e.g., 6, 7, 8, 9, 10, 11, or 12 or more clusters. In one embodiment, the distance between two clusters is greater than the distance between antigenic peptides within a cluster, e.g., 1.5, 2, 2.5, 3, 3.5 times or more the distance between antigenic peptides within a cluster.

[0052] As used herein, an antigenic peptide refers to a peptide having antigenicity or immunogenicity. In particular, the antigenic peptide is derived from a pathogen such as a virus, including but not limited to influenza virus, coronavirus, respiratory syncytial virus, herpes zoster virus, African swine fever virus, malaria parasite, and cytomegalovirus. In addition, the antigenic peptide may be a peptide fragment known in the art that can be used as a subunit vaccine.

[0053] In one embodiment, the antigenic peptide is the S protein receptor binding region (RBD) of SARS-CoV-2 (GenBank: WEU80698.1), the fusion (F) glycoprotein of RSV (GeneID: 1494475), the herpes zoster glycoprotein E (gE) (GenBank: AAY57748), the hemagglutinin (HA) of influenza virus such as influenza A (H1N1) (GenBank: ADC45736.1), the P72 protein of African swine fever virus (GenBank: QNC71679.1), the circumsporiosome protein (CSP) of Plasmodium (GenBank: AAA29526.1) and the cytomegalovirus glycoprotein B (gB) (GenBank: ABQ23592.1). In one embodiment, the antigenic peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 194-200, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with the shown amino acid sequence and is immunogenic.

[0054] As used herein, RBD refers to the receptor binding region of the surface spike glycoprotein of the novel coronavirus (2019-nCoV) (GenBank: WEU80698.1). In one embodiment, the SARS-CoV-2 S protein receptor binding region (RBD) comprises the sequence shown in SEQ ID NO: 194, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the amino acid sequence shown and having immunogenicity.

[0055] In one embodiment, the F glycoprotein comprises the sequence set forth in SEQ ID NO: 195, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% or higher sequence identity thereto and being immunogenic.

[0056] In one embodiment, the gE protein comprises the sequence shown in SEQ ID NO: 196, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% or higher sequence identity to the amino acid sequence shown and is immunogenic.

[0057] In one embodiment, the HA protein comprises the sequence set forth in SEQ ID NO: 197, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% or higher sequence identity thereto and being immunogenic.

[0058] In one embodiment, the P72 protein comprises the sequence shown in SEQ ID NO: 198, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with the amino acid sequence shown and is immunogenic.

[0059] In one embodiment, the CSP protein comprises the sequence shown in SEQ ID NO: 199, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity thereto and being immunogenic.

[0060] In one embodiment, the gB protein comprises the sequence shown in SEQ ID NO: 200, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% or higher sequence identity to the amino acid sequence shown and is immunogenic.

[0061] Herein, the antigen peptide can be connected to the surface of the DNA origami structure through a covalent bond or a non-covalent bond, or connected to the surface of the DNA origami structure through a molecular linker.

[0062] Known methods for linking nucleic acids and proteins are categorized as non-covalent and covalent binding, depending on the form of connection. Non-covalent binding methods primarily include: the interaction between avidin (or streptavidin) and biotin, where protein-nucleic acid connection is achieved through the interaction between biotin-modified (or fused) proteins and nucleic acids coupled to avidin (modified biotin); the binding of metal ions to protein recognition domains, such as nickel ions and polyhistidine, where oligohistidine is fused to proteins and nitrilotriacetic acid (NTA) is modified on nucleic acids, achieving protein-nucleic acid connection under the chelation of polyhistidine-nickel ion-NTA; antigen-antibody affinity and protein-nucleic acid aptamers are also important non-covalent binding methods. Covalent binding is mainly achieved through chemical cross-linking, such as using amino groups on the surface of proteins and alkylthio-modified nucleic acids to form covalent bonds under the action of cross-linkers (such as SSMCC); by modifying the mkhkgs short peptide on the protein and the z-qg (N-benzyloxycarbonyl-L-glutaminylglycine) on the nucleic acid, covalent binding is produced under the action of glutaminase.

[0063] As used herein, site-linking of an antigenic peptide to the DNA origami structure refers to linking the antigenic peptide to the DNA origami structure (e.g., via covalent bonds and / or hydrogen bonds), for example, directly (e.g., covalently or non-covalently) or through any suitable molecule (e.g., a nucleic acid, a peptide, or both) as an intermediate linking molecule, including but not limited to aptamers such as nucleic acid aptamers.

[0064] For example, in one embodiment, a nucleic acid aptamer that specifically binds to an antigenic peptide or a peptide fused to an antigenic peptide can be connected at a desired site of the DNA origami structure, thereby connecting the antigenic peptide to the DNA origami structure. Connecting nucleic acid aptamers at specific sites of the DNA origami structure can be performed using any suitable method known in the art. For example, a specific DNA short chain sequence can be designed, which contains the nucleic acid aptamer sequence. After the DNA origami structure is formed, the nucleic acid aptamer sequence does not pair with the scaffold DNA to form a double strand, but exists as a single strand, thereby achieving connection of the nucleic acid aptamer at the desired site of the DNA origami structure. Optionally, a complementary sequence of the nucleic acid aptamer sequence can be added to form a double-stranded nucleic acid aptamer sequence.

[0065] In one embodiment, the antigenic peptide can be fused to a linker peptide, and then the antigenic peptide and the DNA origami structure are connected by specifically linking the DNA origami domain to the linker peptide. The linker peptide that can be used to fuse with the antigenic peptide can be any suitable peptide, such as those with specific nucleic acid aptamers.

[0066] The antigenic peptide described herein can be directly (e.g., covalently or non-covalently) fused to the connecting peptide (i.e., there is no other molecular sequence between the two, forming an antigenic peptide-connecting peptide or a fusion protein of the reverse order), or can be indirectly fused through a spacer molecule (i.e., forming an antigenic peptide-spacer-connecting peptide or a fusion protein of the reverse order).

[0067] In one embodiment, an adapter molecule can be connected at a site of the DNA origami structure, and the antigenic peptide (for example, at its C-terminus or N-terminus) is fused to a complementary adapter molecule that targets the adapter molecule, thereby connecting the antigenic peptide to the DNA origami structure through the specific interaction between the adapter molecule and the complementary adapter molecule.

[0068] As used herein, "adaptor molecule" and "reciprocal adaptor molecule" refer to a pair of components in a binding pair system, wherein one molecule in the pair specifically binds to the other molecule. Examples of binding pair systems include, but are not limited to, Spycatcher / SpyTag, biotin / avidin, and O6-alkylguanine-DNA alkyltransferase (SNAP-tag) / haloalkane dehalogenase (Halo-tag). Binding can be covalent or non-covalent. "Adapter molecule" refers to one molecule in a pair, and "reciprocal adaptor molecule" refers to the binding partner of the adapter molecule. Thus, in a Spycatcher / SpyTag system, for example, the adapter molecule is SpyCatcher and the reciprocal adaptor molecule is SpyTag, or the adapter molecule is SpyTag and the reciprocal adaptor molecule is SpyCatcher.

[0069] In some embodiments, the adapter molecule is a SpyCatcher and the cooperating adapter molecule is a SpyTag. In some embodiments, the adapter molecule is a SpyTag and the cooperating adapter molecule is a SpyCatcher.

[0070] In some embodiments, the adapter molecule is biotin and the co-matching adapter molecule is avidin. In some embodiments, the adapter molecule is avidin and the co-matching adapter molecule is biotin.

[0071] In some embodiments, the adapter molecule is an O6-alkylguanine-DNA alkyltransferase (SNAP-tag) and the cooperating adapter molecule is a haloalkane dehalogenase (Halo-tag). In some embodiments, the adapter molecule is a haloalkane dehalogenase (Halo-tag) and the cooperating adapter molecule is an O6-alkylguanine-DNA alkyltransferase (SNAP-tag).

[0072] As used herein, a SpyCatcher peptide is any peptide or variant thereof that is capable of reacting with a SpyTag peptide or variant thereof to form an isopeptide bond linkage (Spy reaction). Various SpyCatcher peptides and variants thereof are known in the art, for example, see Zakeri, B., et al. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences, 109(12), E690-E697; Keeble, A Het al. (2017). Evolving accelerated amidation by SpyTag / SpyCatcher to analyze membrane dynamics. Angewandte Chemie International Edition, 56(52), 16521-16525; Keeble, A Het al. (2019). Approaching infinite affinity through engineering of peptide–protein interaction. Proceedings of the National Academy of Sciences, 116(52), 26523-26533.

[0073] The SpyTag and SpyCatcher systems offer highly specific Spy reactions, are relatively small, and can be produced using conventional recombinant expression techniques (e.g., using E. coli as an expression host). The SpyTag / SpyCatcher system is ideal for binding, labeling, or immobilizing proteins because it creates irreversible peptide linkages. SpyTag reacts with SpyCatcher under a wide range of conditions, and the resulting reaction products are highly stable (Zachari et al., 2012, PNAS vol. 109:12, pp. 690-697).

[0074] The antigenic peptide described herein can be directly (e.g., covalently or non-covalently) fused to an adapter molecule (e.g., a Spycatcher peptide) (i.e., there is no other molecular sequence between the two, forming an antigenic peptide-adapter molecule or a fusion protein in the reverse order), or can be indirectly fused through a spacer molecule (i.e., forming an antigenic peptide-spacer-adapter molecule or a fusion protein in the reverse order). Therefore, when referring to an antigenic peptide-Spycatcher fusion protein herein, it covers a fusion protein formed by direct fusion (i.e., antigenic peptide-Spycatcher peptide, or Spycatcher peptide-antigenic peptide) or indirect fusion (i.e., antigenic peptide-spacer-Spycatcher peptide, or Spycatcher peptide-spacer-antigenic peptide) of an antigenic peptide and a Spycatcher peptide in any order.

[0075] The "spacer" or "linker" described herein can be any suitable spacer known in the art for connecting two polypeptides to form a fusion protein, and refers to a peptide composed of amino acids with low hydrophobicity and low charge effect, having a certain length (e.g., 2-16, 5-20, 10-20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, in particular 10-15, preferably 15 amino acids), which, when used in a fusion protein, allows the connected parts to fully unfold and fully fold into their respective native conformations without interfering with each other. Commonly used spacers in the art include, for example, flexible GS-type linkers rich in glycine (G) and serine (S); and rigid PT-type linkers rich in proline (P) and threonine (T). Since GS-type linkers have a more suitable amino acid length, are hydrophobic and ductile, and can make the functional protein have better stability and biological activity, GS-type linkers are preferably used in the present invention, such as GGGGSGGGGSGGGGS (SEQ ID NO: 236).

[0076] In some embodiments, a spacer connects the antigenic peptide to the Spycatcher peptide. In some embodiments, typical amino acid residues for spacers are glycine, serine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, among others.

[0077] In the fusion proteins described herein, the adapter molecule can be located at the C-terminus or N-terminus of the antigenic peptide, i.e., the N-terminus of the adapter molecule is directly or indirectly (e.g., covalently or non-covalently) linked to the C-terminus of the antigenic peptide, or the C-terminus of the adapter molecule is directly or indirectly (e.g., covalently or non-covalently) linked to the N-terminus of the antigenic peptide. In some embodiments, the N-terminus of the adapter molecule is linked to the C-terminus of the antigenic peptide.

[0078] In one embodiment, the N-terminus of the Spycatcher peptide is directly or indirectly (eg, covalently or non-covalently, such as through a spacer) linked to the C-terminus of the antigenic peptide to form a fusion protein.

[0079] In one embodiment, the Spycatcher peptide comprises the amino acid sequence shown in SEQ ID NO:202.

[0080] In one embodiment, the Spytag peptide comprises the amino acid sequence shown in SEQ ID NO:201.

[0081] Attaching an adapter molecule to a site of the DNA origami structure can be performed using any suitable method known in the art, such as using a molecular linker for connecting nucleic acids and proteins. A molecular linker as used herein refers to a chemical moiety capable of covalently linking a protein molecule to a DNA molecule, including, for example, but not limited to, a polymer, a functional group, and the like. A variety of molecular linkers are known in the art that can achieve this purpose, such as those that react with amino groups of proteins and / or amino groups of DNA molecules to form covalent bonds, thereby linking the protein and DNA molecules together.

[0082] In one embodiment, the antigenic peptide is connected to the nucleotide residues of the DNA origami structure via a molecular linker. The molecular linker can be any suitable molecular linker or moiety for connecting nucleotides to amino acids, including but not limited to, in one embodiment, the molecular linkers described herein include the following types: N3 / DBCO; SMCC; SPDP; TCO / tetrazine and HyNic / 4FB. See Beck A et al., Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov. 2017 May; 16(5): 315-337.

[0083] For example, the "N3 / DBCO" refers to a pair of N-hydroxysuccinimide (NHS) ester-modified molecular linkers, wherein the N3 molecular linker (as shown in the following formula) is NHS ester-modified and can react with the NH2 group on the protein, and DBCO (as shown in the following formula) is also NHS ester-modified and can react with the modified NH2 on the DNA, and N3 can undergo a ligation reaction with DBCO, thereby connecting the protein and DNA molecules together.

[0084] In one embodiment, an adapter molecule, such as a SpyTag peptide, is connected to a first single-stranded DNA (e.g., polyT, such as 10-20, 15-20, such as 20 Ts) through a molecular linker to form an adapter molecule-first single-stranded DNA, such as an adapter molecule-polyT, and a second single-stranded DNA (e.g., polyA, such as 10-20, 15-20, such as 15 A's) is included at the site of the DNA origami structure, wherein the second single-stranded DNA is complementary to at least part of the sequence, preferably the entire sequence, of the first single-stranded DNA, thereby connecting the adapter molecule, such as the Spytag peptide, to the DNA origami structure through base pairing of the complementary sequences.

[0085] In one embodiment, the length of the first and second single-stranded DNA can be any suitable length, for example, about 10-50, 10-40, or 10-30 nucleotides, for example, about 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides.

[0086] Herein, the protein can be connected to any suitable site of the DNA via a molecular linker, for example, at the 5' or 3' end, or in the middle of the DNA molecule, preferably at the 5' or 3' end of the DNA. These connections are within the knowledge of those skilled in the art.

[0087] In particular, in one embodiment, an azide-modified adaptor molecule, such as a SpyTag peptide, is connected to a first single-stranded DNA (e.g., polyT) containing DBCO via an N3 / DBCO molecule linker, and a second single-stranded DNA (e.g., polyA) is contained at the site of the DNA origami structure, wherein the second single-stranded DNA is complementary to the first single-stranded DNA in at least a portion of its sequence, preferably in its entirety, thereby connecting the adaptor molecule, such as the Spytag peptide, to the DNA origami structure through base pairing of the complementary sequences. Accordingly, the antigenic peptide can be connected to a complementary adaptor molecule, such as a Spycatcher peptide, to form a fusion protein, thereby connecting the antigenic peptide to the DNA origami structure through the interaction between the adaptor molecule and the complementary adaptor molecule.

[0088] In one embodiment, in one aspect, a method for preparing the subunit vaccine based on the DNA origami structure of the aforementioned aspect is provided, comprising:

[0089] (a) Provide a DNA origami structure,

[0090] (b) attaching a plurality of antigenic peptides to the DNA origami structure, respectively, wherein the antigenic peptides are distributed in clusters on the DNA origami structure, the number of antigenic peptides in each cluster is 2-6, preferably 5, and the distance between the antigenic peptides is about 5-20 nm (preferably the antigenic peptides are uniformly distributed, preferably with a distance of about 10 nm), and the number of clusters per DNA origami structure is 1-20 (e.g., 6), and

[0091] (c) Harvesting the formed DNA origami-based subunit vaccine.

[0092] The structural features of the DNA origami structure are as described above. Those skilled in the art can design and prepare DNA origami structures with desired features (e.g., shape, specific sites, or containing specific linkers or nucleotide sequences) and connect antigen peptides to the desired sites of the DNA origami structure according to methods known in the art. See, for example, Paul WK Rothemund, Folding DNA to create nanoscale shapes and patterns, Nature V440: 297-302 (16 March 2006); RP Goodman et al., Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication, Science, V310: 1661-1664 (9 December 2005).

[0093] In one aspect, a pharmaceutical composition is provided, comprising the subunit vaccine of the aforementioned aspect or the subunit vaccine prepared according to the aforementioned method, and a pharmaceutically acceptable carrier.

[0094] A "pharmaceutically acceptable carrier" refers to a substance that facilitates administration and absorption of an active substance into a subject and can be included in the compositions of the present invention without causing significant toxic side effects in the patient. Pharmaceutically acceptable carriers (vehicles) suitable for use in the present invention are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, ed. Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st edition (2005) describes compositions and formulations suitable for drug delivery.

[0095] Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and coloring agents, etc. Those skilled in the art will appreciate that other pharmaceutical carriers may be used in the present invention.

[0096] The term "pharmaceutical composition" as used herein refers to a pharmaceutical product formulated to meet specific dosage requirements for the treatment or prevention of a disease and intended for use by a patient. The pharmaceutical composition may contain the subunit vaccine described herein, as well as other pharmaceutical excipients and tools.

[0097] The "excipients" mentioned in this article are pharmaceutical excipients, which refer to substances other than active ingredients used in the production of drugs and the preparation of prescriptions, which have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. In addition to their purpose of excipients, serving as carriers or improving stability, they can also have important functions such as solubilization, dissolution assistance, and sustained-release.

[0098] The medicament or pharmaceutical composition herein can be formulated into a dosage form suitable for any suitable administration route, such as intravenous, subcutaneous, parenteral, oral, intraperitoneal, etc., such as tablets, powders, solutions, etc. In one embodiment, the medicament or pharmaceutical composition of the present invention can be formulated into a form suitable for intravenous administration.

[0099] In one aspect, the present invention provides the use of the subunit vaccine of the aforementioned aspect or the subunit vaccine prepared according to the method of the aforementioned aspect in the preparation of a medicament for preventing or treating a pathogen, such as a viral infection, or provides the use of the subunit vaccine of the aforementioned aspect or the subunit vaccine prepared according to the method of the aforementioned aspect for preventing or treating a pathogen, such as a viral infection. In addition, a method for preventing or treating a pathogen, such as a viral infection, in a subject is provided, comprising administering to the subject a therapeutically effective amount of the subunit vaccine of the aforementioned aspect or the subunit vaccine prepared according to the method of the aforementioned aspect.

[0100] As used herein, a "patient" or "subject" refers to an organism suffering from or susceptible to a disease or condition that can be treated by administering a DNA origami-based subunit vaccine as provided herein. Non-limiting examples include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient or subject is a human.

[0101] For a given pathogen, such as a virus, a subunit vaccine containing the corresponding antigenic peptides from the pathogen or antigenic peptides that are immunogenic to the pathogen and arranged in clusters in a DNA origami structure as described herein can be administered to a subject to prevent or treat infection by the pathogen or related diseases or conditions.

[0102] In one embodiment, the pathogen is selected from viruses such as influenza virus, coronavirus, respiratory syncytial virus, herpes zoster virus, African swine fever virus, malaria parasite and cytomegalovirus.

[0103] The word "or" is intended to include "and" unless the context indicates otherwise.

[0104] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist.

[0105] As used herein, the term "about" refers to a numerical range that includes a specific numerical value, and a person skilled in the art can reasonably believe that it is similar to a specific numerical value. In certain embodiments, the term "about" refers to within the standard error of measurement generally accepted in the art. For example, in certain embodiments, about refers to + / - 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or lower of a specific numerical value.

[0106] As used herein, the term "approximately equal" means that two values ​​are approximately equal, for example, differing by no more than + / - 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less.

[0107] As used herein, when a specific value or ratio is listed for a feature in the specification, a range consisting of any two of the values ​​or ratios is also included. For example, when the values ​​1, 2, 3, and 4 are listed, 1-2, 1-3, 1-4, 2-3, 2-4, and 3-4 are also included. DETAILED DESCRIPTION

[0108] The following examples further illustrate the specific embodiments of the present invention, but the embodiments of the present invention are not limited thereto. The methods used in the following examples are conventional methods unless otherwise specified.

[0109] Materials and methods

[0110] Reagents and materials. Oligonucleotides used for folding ICO (icosahedron) were synthesized by Beijing Ruibio Biotech Co., Ltd. (Beijing, China). Genomic DNA of M13mp18 phage was obtained from Beijing Intell Nanomedicine (Beijing, China). DBCO-PolyT (20×T, SEQ ID NO: 235), SpyTag-N3, Cy5.5-SpyTag, and FITC-SpyTag were synthesized by China Peptides Co., Ltd. (Shanghai, China), where Spytag is shown as SEQ ID NO: 201. The RBD-specific BCR expression plasmid pRRL-EuB29 COVA2-15 IgGTM.BCR.GFP.WPRE (PJM Brouwer et al., Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection; Cell, 2021) was kindly provided by Professor Marit J. van Gils and Professor Rogier W. Sanders of the Department of Medical Microbiology, University of Amsterdam. The SARS-CoV-2 pseudovirus neutralization test kit (GenScript, China, catalog: #L02087A), including the SARS-CoV-2 pseudovirus Luc reporter and hACE2-expressing HEK-293 cells, was purchased from GenScript (Nanjing, China).

[0111] Antibodies used for flow cytometry were as follows: anti-His Tag APC (BioLegend, USA, Cat:#362605), anti-human IgG (G18-145) APC (BD, USA, Cat:#550931), anti-mouse CD3 APC (BioLegend, Cat:#100312), anti-mouse CD19 APC (BioLegend, Cat:#115512), anti-mouse CD138 APC (Biolegend, Cat:#142506), anti-mouse CXCR5 APC (Biolegend, Cat:#145506), anti-mouse CD103 APC (Biolegend, Cat:#121414), anti-mouse CD8 FITC (Biolegend, Cat:#100706), anti-mouse CD11C FITC (Biolegend, Cat:#117306), anti-mouse CD95 (Fas) FITC (Biolegend, Cat:#152606), anti-mouse IgG FITC (Biolegend, Cat:#406001), anti-mouse CD4 PE (Invitrogen, USA, Cat:#12-0042-85), anti-mouse CD44 PE (Biolegend, Cat:#103008), anti-mouse CD80 PE (Biolegend, Cat:#104708), anti-mouse CXCR3 PE (Biolegend, Cat:#126505), anti-mouse CD103 PE (Biolegend, Cat:#121406), anti-mouse CD21 / CD35 PE (Biolegend, Cat:#123410), anti-mouse CD69 PE (Biolegend, Cat:#104508), anti-mouse CD44 PE (Biolegend, Cat:#103008), anti-mouse CD86 PE / Cyanine7 (Biolegend, Cat:#105014), anti-mouse IFNγ PE / Cyanine7 (Biolegend, Cat:#505826), anti-mouse F4 / 80 PE / Cyanine7 (Biolegend, Cat:#123114), anti-mouse CD279 (PD-1) PE / Cyanine7 (Biolegend, Cat:#109109), anti-mouse / human GL7 PE / Cyanine7 (Biolegend, Cat:#144619), anti-mouse EpCAM PE / Cyanine7(Biolegend,Cat:#118215), anti-mouse CD38 PE / Cyanine7 (Biolegend, Cat:#102718), anti-mouse IL2 PE / Cyanine7 (Biolegend, Cat:#503831), anti-mouse IL4 PE / Cyanine7 (Biolegend, Cat:#504117), anti-mouse TNF-α PE / Cyanine7 (Biolegend, Cat:#506323), anti-mouse CD62L PE / Cyanine7 (Biolegend, Cat:#104418), anti-mouse CD8a PE / Cyanine7 (Biolegend, Cat:#100722), anti-mouse / human B220APC / Cyanine7 (Biolegend, Cat:#103223), anti-mouse CD4 PerCP(Biolegend,Cat:#100432). ,

[0112] Protein expression and purification. Expression and purification of SpyCatcher-RBD (SpyCatcher is shown in SEQ ID NO: 202; RBD is residues 319-541, as shown in SEQ ID NO: 194) in HEK-293 cells were completed at AtaGenix Laboratories Co., Ltd. (Wuhan, China). Briefly, the 8×His-tagged SpyCather-RBD DNA sequence (Figure 7a) was cloned into the pATX2 vector and transfected into HEK-292 cells. After 7 days, the culture medium and soluble fraction were collected, and the His-tagged target protein was purified by affinity Ni-NTA, then eluted with Tris buffer containing imidazole and concentrated. The purified His-tagged protein was identified by Coomassie blue staining and Western blotting. Protein concentration was determined using the BCA method.

[0113] Preparation and purification of ICO. ICO design (115.5 bp side length) was performed using the algorithm framework DAEDALUS (http: / / daedalus-dna-origami.org). The sequence information of the staple strand of ICO is provided in Table 1. The sequence information of the capture strand in the ICO-RBD nanovaccine (ICO-RBD-6c×5-10 nm) is shown in Table 2. DNA origami was prepared by the following one-pot reaction: 20 nM M13mp18 scaffold was mixed with 100 nM of the staple strand or capture strand in 1× TAE-MgCl2 solution. 2+The DNA was self-assembled in a 5% PBS buffer (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, 12.5 mM MgCl2, pH = 8.0). The mixture was then self-assembled using a thermal cycler (T100, Bio-rad) through an annealing program: 95°C for 5 minutes, 1°C / 5 minutes to 80-75°C, 1°C / 20 minutes to 75-30°C, and 1°C / 10 minutes to 30-25°C. Fresh DNA origami was purified three times using a 300KD centrifugal filter (RealTimes, China, Cat: #RT-VS0152-5) to remove excess short chains and capture chains. The origami was quantified by NanoDrop 2000 (Thermo Fisher Scientific, USA) and stored at 4°C for subsequent analysis. The origami was characterized using 0.6% agarose gel electrophoresis and imaged by TEM (JEM-1400) or AFM (Bruker, Multimode 8).

[0114] Table 1: Short chain sequences of ICOs

[0115] Table 2: Sequence information of the capture strand of ICO-RBD-6c×5-10nm

[0116] Antigen connection of ICO. SpyTag-N3 and DBCO-PolyT were mixed in a molar ratio of 1:1 and reacted at 4°C overnight to obtain SpyTag-PlyT conjugate. Purified ICO was mixed with SpyTag-PolyT conjugate on ICO at a molar ratio of Poly T:PolyA of 5:1. PolyT-PolyA hybridization was performed in a thermal cycle: 1°C / 5 minutes to 45-25°C, 5 cycles, and finally kept at 4°C. The product (ICO-SpyTag) was purified 3 times with a 300KD centrifugal filter (RealTimes, Cat:#RT-VS0152-5) to remove excess SpyTag-PolyT conjugate. Then, SpyCatcher-RBD protein was added to ICO-SpyTag at a molar ratio of SpyCatcher:SpyTag of 3:1. The mixture was incubated at room temperature for 3 hours, and the excess Spycatcher-RBD protein was removed by ultrafiltration.

[0117] Analysis of the efficiency of ICO-linked antigens. The loading efficiency was quantified using ratiometric absorbance measurements. In detail, the concentration of DNA origami was measured by absorbance at 260 nm using a NanoDrop 2000 spectrophotometer with an extinction coefficient of 10.9 × 10 7 M -1 cm -1 Antigen concentrations were quantified using the BCA standard curve method. Antigen coverage efficiency was defined as the percentage of the measured protein concentration in the nanovaccine relative to the theoretical concentration in the nanovaccine. Statistical data were obtained from at least three independent experiments.

[0118] Preparation of B-RBD cells. The expression plasmid pRRL EuB29COVA2-15IgGTM.BCR.GFP.WPRE and the packaging plasmid mixture (mixture = pMDL:pVSV-g:pRSV-Rev = 5:3:2) were co-transfected into HEK-293T cells at a ratio of 1:1 using lipofectamine 3000 (Invitrogen, USA, Cat:#L3000015). After 48 hours, the cell supernatant was collected and filtered (0.45 μm), and then 50 μl of concentrated viral supernatant was added to pre-seeded Ramos B cells (2×10 6 7 days after transfection, GFP was detected by FACS (BD Aria III). + IgG + The cell population (B-RBD cells) was sorted.

[0119] Cellular affinity determination of ICO-RBD and B-RBD. B-RBD cells were grown in RPMI 1640 medium (Gibco, Cat: #C11875500CP) containing 10% fetal bovine serum (Gibco, Cat: #10091148) and 1% penicillin-streptomycin (Biological Industries, Cat: #03-031-1B). Cells (1×10 6 ) were then suspended in PBS and incubated with 0.5 μg / mL SpyCatcher-RBD protein or different ICO-RBD nanovaccines at the same protein concentration at 4°C for 1, 2, 4, 6, 8, and 10 minutes. The cells were then washed three times with PBS and incubated with anti-His antibody at 4°C for 40 minutes. The cells were washed twice with PBS and SpyCatcher-RBD protein or ICO-RBD nanovaccines bound to B-RBD cells were detected by flow cytometry.

[0120] B cell activation assay. B-RBD cells (1×10 74 μM Fluo-4AM (Cat:#F14217, Invitrogen, USA) was added to 2×10 cells / mL) at 37°C for 20 minutes. The cells were washed twice with HBSS buffer (Solarbio, China, Cat:#H1025) and then incubated at 37°C for 30 minutes. The washed and collected cells were resuspended in PBS, and the intracellular calcium changes induced by the nanovaccine were detected by flow cytometry (excitation / emission: 495 nm / 518 nm). After 35 s fluorescence baseline recording, 50 μL SpyCatcher-RBD protein or ICO-RBD nanovaccine (5 μg / mL RBD) was added to 2×10 6 Cells were plated at 50 cells / mL and fluorescence was monitored for 3-5 minutes. At each time point, 50 μL of PBS was added to record the fluorescence, which was used as a normalization standard.

[0121] Animal vaccination. All animal experiments were performed according to animal use protocols approved by the Committee for the Ethics of Animal Experiments, the Institutional Animal Care and Use Committee of the National Center for Nanoscience and Technology.

[0122] Six-week-old female BALB / c mice were randomly divided into 8 groups: PBS (control), ICO, RBD, ICO+RBD (physical mixture), ICO-RBD-1c×5-10nm, ICO-RBD-1c×5-40nm, ICO-RBD-6c×1-10nm, and ICO-RBD-6c×5-10nm. At weeks 0 and 3, BALB / c mice (n=8) were immunized intramuscularly with two doses of 50μL nanovaccine containing 1mg of SpyCatcher-RBD protein mixed with 50μL Addavax (Invivogen, USA, Cat:#vac-adx-10). Serum samples were collected at weeks 2, 5, 8, 11, and 14, and RBD-specific IgG titers were measured using ELISA. Sera collected at weeks 5 and 14 were analyzed using a pseudovirus neutralization assay. All mice were euthanized at week 14 and memory immune cells were evaluated.

[0123] ELISA. Spycatcher-RBD protein was diluted to 5 μg / mL using ELISA coating buffer (Solarbio, Cat:#C1055) and coated onto 96-well ELISA plates (Thermo Fisher, USA, Cat:#442404) at 4°C overnight. The washed plates were blocked with 5% skim milk in PBS for 2 hours. Serum was serially diluted and added to each well and incubated at room temperature for 2 hours. After washing the plates three times with PBS containing 1% Tween-20 (PBST), goat anti-mouse IgG HRP antibody (Proteintech, USA, Cat:#SA00001-1) was diluted 1:5000 in 5% skim milk and incubated at 37°C for 1 hour. The plates were washed with PBST and then developed with 3,3',5,5'-tetramethylbenzidine (TMB) (Solarbio, Cat:#PR1200). The color reaction was stopped by ELISA stop solution (Solarbio, Cat: # C1058) and the absorbance at 450 nm was read by a microplate reader (BioTEK, USA, Synergy H1). The data were analyzed by nonlinear regression to calculate the endpoint titer.

[0124] Pseudovirus neutralization assay. Pseudovirus neutralization assay was performed according to the manufacturer's instructions. Briefly, serum samples and positive control (ACE2-Fc protein) were serially diluted and then mixed with revived SARS-CoV-2 pseudovirus at a volume of 1:1 for 1 hour at room temperature, where the virus titer was 3×10 4 TCID 50 / mL. The mixture was added with hACE2 (6×10 5 / mL) in HEK-293 cells expressing SARS-CoV-2 and cultured at 37°C in a 5% CO2 incubator for 48 hours. Luciferase activity was analyzed using a luciferase assay system (GenScript, Cat: #L00877C). Relative luminescence units (RLU) were normalized to the relative luminescence units of cells infected with SARS-CoV-2 pseudovirus in the absence of serum. Neutralization titer (ID 50 The serum dilution that inhibited infectivity by 50% was determined.

[0125] Analysis of memory T cells, antigen-specific MBCs, and LLPCs. Splenocytes from mice were obtained at week 14 and prepared as single-cell suspensions. For RBD-specific MBC analysis, Cy5.5-SpyTag was conjugated to SpyCatcher-RBD protein to prepare the "Cy5.5-RBD" fluorescent probe. The cells were incubated with Cy5.5-RBD at 4°C for 1 hour and washed 3 times with PBS, then fixed and perforated. Antigen-specific MBCs in splenocytes were analyzed using B220, IgG, and CD38 antibodies. For LLPC analysis, splenocytes and bone marrow cells (from femurs and tibias) were obtained, and red blood cells were removed to obtain single-cell suspensions. FITC-RBD probes were prepared according to the above method. The cells were mixed with FITC-RBD at 4°C for 1 hour and then washed 3 times with PBS. B220, CD138, EpCAM, and CXCR3 antibodies were used to identify antigen-specific LLPCs. Central memory T cells and effector memory T cells in splenocytes were analyzed by flow cytometry after staining with CD3, CD8, CD44, and CD62L antibodies.

[0126] Intracellular cytokine staining assay and TRM analysis. The spleen and lungs of mice were collected at 3 weeks and 6 weeks. The spleen was ground and passed through a 40 μm cell strainer to obtain a single cell suspension. The lungs were cut into pieces and treated with the prepared digestion buffer (0.05% trypsin solution, 1 mg / mL DNase and 1 mg / mL collagenase D) at 37°C and 120 rpm for 30 min. The cells were stimulated with SpyCatcher-RBD protein overnight, and then the cells were collected and stained with CD3, CD4 and CD8 antibodies. After surface marker staining, the cells were fixed and perforated, and the cells were stained with IFN-γ, IL-2, TNF-α and IL-4 antibodies, respectively. CD3 was analyzed using flow cytometry. + CD8 + T cells and CD3 + CD4 + IFN-γ in T cells + cells, IL-2 + cells, TNF-α + cells and IL-4 + The proportion of cells and TRMs in the lungs were analyzed by flow cytometry after staining with CD8, CD69, and CD103 antibodies.

[0127] DC and macrophage studies. Six-week-old female BALB / c mice were immunized intramuscularly with 50 μL of Cy5.5-labeled SpyCatcher-RBD protein or a Cy5.5-labeled nanovaccine containing 1 mg of SpyCatcher-RBD protein mixed with 50 μL of Addavax. Twelve hours later, inguinal lymph nodes were obtained and then subjected to fluorescence imaging. Single cell suspensions were obtained after trituration. DC (CD11C + ), resident DC (CD103 - CD11C + 、CD4 + CD11C + or CD8a + CD11C + ), migrated DCs (CD103 + CD11C + ), follicular DC (CD21 / CD35 + CD11C + ), mature DC (CD11C + CD80 + 、CD11C + CCD86 + ) and macrophages (F4 / 80 + ) in Cy5.5 + cell.

[0128] GC B, T FH Flow cytometric analysis of GC B cells and T cells FH For cell response analysis, 6-week-old female BALB / c mice were randomly divided into three groups: PBS, RBD, and ICO-RBD nanovaccines. Inguinal lymph nodes were harvested at 1, 2, and 3 weeks after immunization. Single cell suspensions were obtained by grinding and sieving. GC B cells (CD19 + B220 + CD95 + GL7 + ), T FH cells (CD4 + CXCR5 + PD-1 + ) and plasma cells (CD44 + CD138 + ) percentage.

[0129] Statistical analysis

[0130] Details of the statistical analysis, including sample size and mean ± standard error of the mean (SEM) or mean ± standard deviation (SD), are provided in the figure legends. Statistical analysis was performed using Prism 8 (GraphPad) on data from at least three independent experiments. For multiple group comparisons, one-way ANOVA and Tukey's test were used.

[0131] Example 1: Design and construction of ICO-RBD nanovaccine

[0132] ICO was assembled by slowly annealing the genomic DNA of the M13mp18 phage (SEQ ID NO: 203) as a template chain with multiple short chains and capture chains (Tables 1 and 2). The successful assembly of ICO was demonstrated by the migration of band positions in agarose gel electrophoresis analysis (Figure 1c). Under observation using negative staining transmission electron microscopy (TEM) and atomic force microscopy (AFM), ICO exhibited a uniform hollow polyhedral structure with a diameter of approximately 80-90 nm (Figures 1d-e). The hydrophilic diameter detected using dynamic light scattering (DLS) was 93 nm (Figure 1f), which is close to the hydrophilic diameter of SARS-CoV-2.

[0133] Next, the RBD antigen (SEQ ID NO: 194) was modified onto ICO using an “engraving printing” strategy (Figure 1a). Briefly, the 5' end of the short chain at the designed position was extended with PolyA to form a capture chain, and even if the number of capture chains was increased to 60, the hanging PolyA capture chain did not interfere with origami assembly (Figure 6a). Next, the C-terminal azide-modified SpyTag peptide glue (SpyTag-N3) and the 5'-terminal dibenzocyclooctene (DBCO)-modified PolyT (DBCO-PolyT) were covalently linked by click chemistry to form a SpyTag-PolyT conjugate. The SpyTag peptide was then engraved onto ICO through DNA complementary hybridization between the PolyT in the SpyTag-PolyT conjugate and the protruding PolyA in the capture chain (Figure 6b). Finally, the fusion protein of SpyCatcher and RBD (N-SpyCatcher-RBD-C) (Figure 7a-c) was printed onto ICO through spontaneous covalent isopeptide bond formation between SpyCatcher and SpyTag, ultimately achieving site-specific modification of RBD antigens on the ICO surface. We designed and constructed five different ICO-RBD nanovaccines, with 12, 24, 36, 48, and 60 RBD antigens modified on the surface, respectively. As the number of modified RBD antigens increased, the electrophoretic rate of ICO-RBD decreased (Figure 1g), indicating successful protein modification. DNA and protein concentration measurements confirmed that when the number of RBD antigens was in the range of 12-60, the modification efficiency of RBD antigens in ICO-RBD reached over 85% (Figure 1h) due to the mild conditions and rapid reaction characteristics of click chemistry and peptide glue technology. In addition, the site-specific conjugation based on peptide glue technology ensured the uniform outward orientation of RBD antigens on the ICO-RBD nanovaccine, which is conducive to antigenic immune stimulation of B cells.

[0134] Example 2: Effects of ICO-RBD Nanovaccines with Different Surface Antigen Patterns on B Cell Triggering

[0135] Next, we investigated the relationship between surface antigen patterns and the ability of nanovaccines to activate B cells. Given the cluster distribution characteristics of RBD antigens in real SARS-CoV-2, the surface RBD patterns in ICO-RBD nanovaccines were also designed as clusters, but several parameters of the surface RBD antigen patterns in ICO-RBD nanovaccines were controlled, including antigen spacing, antigen copy number within clusters, and cluster number (Figure 2a). Different ICO-RBD nanovaccines were referred to as ICO-RBD xc×nd nm, where x represents the number of clusters, n represents the number of antigen copies within a cluster, and d represents the antigen spacing within a cluster. To evaluate the ability of nanovaccines to activate B cells, we generated B cells (B-RBD cells) expressing BCRs of the previously described RBD-targeting monoclonal neutralizing antibody COVA2-15 [Brouwer PJM et al. Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection. Cell. 2021; 184: 1188-200.e19; Brouwer PJM, et al. Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability. Science (New York, NY). 2020; 369: 643-50] and measured cell binding and Ca 2+ induced by different ICO-RBD nanovaccines in vitro. 2+ Inflow (Figure 2b).

[0136] First, we established an ICO-RBD nanovaccine, which has only one RBD cluster with 5 RBD copies within the cluster, but the RBD spacing varies from 10 to 40 nm (Figure 2c and Figure 8a-b). As the RBD spacing decreases, the affinity between the ICO-RBD nanovaccine and B-RBD cells gradually increases (Figure 2d). At the same time, the ICO-RBD nanovaccine triggers the Ca 2+ The ability of influx is also negatively correlated with the RBD distance (Figure 2e). When the RBD distance reaches 40 nm, ICO-RBD nanovaccine activates B-RBD cell Ca 2+ The ability to enter was similar to that of soluble RBD antigens (Figure 2e). These results indicate that clustered distribution does enhance B cell activation compared to soluble RBD antigens, but strong B cell activation requires sufficiently close antigen spacing. When the antigen spacing is too large, these RBD antigens lose their clustered distribution characteristics and resemble RBD monomers.

[0137] Next, we attempted to determine the effect of the number of antigen clusters in the ICO-RBD nanovaccine on B cell activation. The number of antigen copies within the cluster was fixed at 5, the antigen spacing was fixed at 10 nm, but the number of clusters was controlled at 1-12 (Figure 2f and Figure 8c-d). As shown in Figure 2g, the increase in the number of antigen clusters increased the binding ability between the ICO-RBD nanovaccine and B-RBD cells, but when the number of antigen clusters reached 6, the binding ability reached saturation, which was indicated by the similar cell binding of ICO-RBD-6c×5-10nm and ICO-RBD-12c×5-10nm. Among the ICO-RBD nanovaccines with different numbers of antigen clusters, ICO-RBD-6c×5-10nm was also the most effective in activating B-RBD cell Ca. 2+ The best candidate for influx (Figure 2h). In general, the surface multi-cluster pattern is close to the surface of SARS-CoV-2 [Ke Z, et al. Structures and distributions of SARS-CoV-2 spike proteins on intact virions. Nature. 2020; 588: 498-502], which is conducive to the immune stimulation of nanovaccines.

[0138] To further investigate the role of cluster antigen copies on B cell responses, we programmed several ICO-RBD-6c×n-10nm nanovaccines, with clusters containing 1 to 5 antigen copies (Figures 8e-f and 4a). As shown in Figures 9b-c, the number of antigen copies within the cluster was also an important parameter influencing the immunostimulatory capacity of the ICO-RBD nanovaccine. Interestingly, despite being identical to SARS-CoV-2, the ICO-RBD-6c×3-10nm nanovaccine with 3 antigen copies within the cluster was not the most effective. Furthermore, as the number of antigen copies within the cluster increased, the effectiveness of the nanovaccine continued to improve, highlighting the importance of rational artificial vaccine design. In summary, through screening and evaluation of different parameters, the ICO-RBD-6c×5-10nm nanovaccine was identified as the optimal ICO-RBD nanovaccine, and the following in vivo studies were conducted.

[0139] Example 3: ICO-RBD nanovaccine induces a strong humoral immune response in vivo

[0140] To evaluate the immunogenicity of the ICO-RBD nanovaccine in vivo, 6-week-old BALB / c mice were intramuscularly immunized twice with different ICO-RBD nanovaccines containing 1 μg of RBD and formulated with AddaVax adjuvant (Figure 3a). Phosphate-buffered saline (PBS), RBD monomer, ICO, and RBD+ICO (physical mixture) were used as controls, respectively. As detected by enzyme-linked immunosorbent assay (ELISA), the serum titers of SARS-CoV-2 RBD-specific immunoglobulin G (IgG) in the RBD and ICO-RBD-6c×5-10nm groups reached 148 and 1738, respectively, at week 2 after priming (Figure 3b). After boosting, the RBD-specific IgG titers elicited by the ICO-RBD-6c×5-10nm nanovaccine were higher than 10 5 , while RBD monomer, RBD+ICO and other ICO-RBD nanovaccines including ICO-RBD-1c×5-10nm, ICO-RBD-1c×5-40nm and ICO-RBD-6c×1-10nm only produced 10 3 -10 4 We monitored the RBD-specific IgG titer for 14 weeks, and the time-titer curve showed that the RBD-specific IgG titer of the ICO-RBD-6c×5-10nm group peaked at week 8 and only slightly decreased at week 14 (Figure 3d). However, the RBD-specific IgG titer of the other groups peaked at week 5 and dropped sharply at week 14 (Figure 3d). Sera collected at weeks 5 and 14 were used in pseudovirus neutralization assays to analyze neutralizing antibodies, such as the inhibitory dilution value (ID) that achieved 50% neutralization. 50 ) is shown. Consistent with the RBD-specific IgG titer results, ICO-RBD-6c×5-10nm induced ID 50 The value was more than 100 times that of the RBD group (Figure 3e-f). In summary, the ICO-RBD-6c×5-10nm nanovaccine was highly immunogenic compared to the RBD monomer.

[0141] Next, to evaluate whether the ICO-RBD nanovaccine could induce long-lasting immunity, several memory immune cells, including memory B cells (MBCs), long-lived plasma cells (LLPCs), effector memory T cells, and central memory T cells, were evaluated at week 14 [Akkaya M, et al. B cell memory: building two walls of protection against pathogens. Nature reviews Immunology. 2020; 20: 229-38; Liu X, et al. Heterogeneous plasma cells and long-lived subsets in response to immunization, autoantigen and microbiota. Nature immunology. 2022; 23: 1564-76]. Compared with RBD monomers, ICO-RBD-6c×5-10nm nanovaccine induced more RBD-specific IgG. + MBCs (Figure 3g). The proportion of LLPCs in the ICO-RBD-6c×5-10nm group in RBD-specific bone marrow plasma cells and spleen plasma cells was also higher than that in the RBD group (Figure 3h-i and Figure 10a-b). In addition, compared with mice in the RBD group, although effector memory T cells were not significantly induced (Figure 10c), the central memory T cells in the spleen cells of mice immunized with the ICO-RBD-6c×5-10nm nanovaccine were significantly increased (Figure 3j). Based on the above results, the DNA origami-based nanovaccine induced higher neutralizing antibodies against RBD and more RBD-specific memory immune cells than the RBD monomer, which can provide a long-lasting protective humoral response.

[0142] ICO-RBD nanovaccine induces T cell immune responses in vivo

[0143] Many CD4s in RBD + and CD8 +T cell epitopes, which are also critical for viral defense and clearance [Jiang S, et al. Identification of a promiscuous conserved CTL epitope within the SARS-CoV-2 spike protein. Emerging microbes & infections. 2022; 11: 730-40; Tarke A, et al. SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron. Cell. 2022; 185: 847-59. e11]. Next, to measure the T cell responses induced by the ICO-RBD nanovaccine (ICO-RBD-6c×5-10nm), we performed intracellular cytokine staining assays to analyze different T cell populations in the spleen and lungs at weeks 3 and 6, respectively. As shown in Figures 4a-d, in spleen or lung tissue, ICO-RBD nanovaccine induced more RBD-specific CD8 cytokines expressing interferon-γ (IFN-γ), interleukin (IL)-2, and tumor necrosis factor-α (TNF-α) than RBD monomer. + and CD4 + T cells, indicating that the ICO-RBD nanovaccine induced a strong type 1 T helper cell (Th1) biased immune response in vivo. After priming and boosting immunization, few CD4 T cells expressing IL-4 were induced in the three groups. + T cells, indicating that Th2-biased immune cells were not induced (Figure 4e-f). Tissue-resident memory T cells (TRMs) rapidly mobilize innate and adaptive immunity in the lungs to achieve an antiviral state [Schenkel JM, et al. T cell memory. Resident memory CD8 T cells trigger protective innate and adaptive immune responses. Science (New York, NY). 2014; 346: 98-101]. As shown in Figure 4g, RBD monomers did not induce obvious TRMs in lung tissue, but ICO-RBD nanovaccines produced more TRMs in the lungs after boosting immunity. These results indicate that in addition to B cell responses, ICO-RBD nanovaccines can also induce strong T cell immune responses.

[0144] ICO-RBD nanovaccine induces antigen presentation and germinal center responses in draining lymph nodes

[0145] To further elucidate how ICO-RBD nanovaccine is recognized and processed by the host immune system and participates in the immune response, RBD monomers and ICO-RBD nanovaccines (ICO-RBD-6c×5-10nm) were labeled with fluorescein Cy5.5. Twelve hours after intramuscular injection, inguinal lymph nodes were isolated. As shown in Figure 5a, more ICO-RBD nanovaccines accumulated in the inguinal lymph nodes compared with RBD monomers. Detailed analysis of vaccine uptake by antigen-presenting cells showed that dendritic cells (DCs, CD11C + ), mature DCs (CD11C + CD80 + and CD11C + CD86 + ) and macrophages (F4 / 80 + ) captured more ICO-RBD nanovaccines than RBD monomers (Figure 5b and Figure 11a-b). + CD8a + ) and CD103 + Migrating DCs (CD11C + CD103 + There was no significant difference between the captured ICO-RBD nanovaccine and RBD monomer (Figure 11c-d). However, in CD4 + resident DCs (CD11C + CD4 + ) and CD103 - resident DCs (CD11C + CD103 - ) and follicular DCs (CD11C + CD21 / CD35 + ), the capture difference was obvious (Figure 5b and Figure 11e). These results indicate that ICO-RBD nanovaccines are actively drained into lymph nodes and are more easily captured by resident and follicular DCs than monomeric RBD, which may be closely related to the stronger T cell immunity induced by ICO-RBD nanovaccines.

[0146] Compared with soluble vaccines, nanoparticle vaccines are more easily captured by DCs and macrophages, thereby promoting the activation of T follicular helper cells (T FH) and B cells, which is required for long-term effective vaccination to drive antibody-mediated humoral immunity [Kelly HG, et al. Self-assembling influenza nanoparticle vaccines drive extended germinal center activity and memory B cell maturation. JCI insight. 2020; 5; Zhang YN, et al. Nanoparticle Size Influences Antigen Retention and Presentation in Lymph Node Follicles for Humoral Immunity. Nano letters. 2019; 19: 7226-35]. To assess the germinal center response, we examined T cells in the inguinal lymph nodes at 1, 2, and 3 weeks after priming. FH cells (CD4 + PD1 + CXCR5 + ), germinal center (GC) B cells (CD19 + B220 + GL7 + CD95 + ) and plasma cells (CD44 + CD138 + )(Figure 5c). Although RBD monomers induced a significant T FH T cells, GC B cells, and plasma cells were induced by ICO-RBD nanovaccine (Figure 5d). More importantly, ICO-RBD nanovaccine maintained T cell proliferation for a longer period of time than RBD monomer. FH The ICO-RBD nanovaccine showed a high frequency of RBD cells, GC B cells, and plasma cells (Figure 5d). As a result, the serum titer of RBD-specific IgG in the ICO-RBD nanovaccine group gradually increased from week 1 to week 3, while the serum titer in the RBD group remained stable (Figure 12a-b). In summary, the ICO-RBD nanovaccine was more effectively captured by antigen-presenting cells than the RBD monomer and stimulated a stronger germinal center response, thereby achieving more effective antigen presentation, TB crosstalk, and B cell maturation.

[0147] Example 4: Construction of ICO-HA, ICO-P72, ICO-CSP, and ICO-gB Nanovaccines and In Vivo Evaluation

[0148] The "engraving printing" strategy used in Example 1 was used to modify ICO with influenza A antigen HA (SEQ ID NO: 197), African swine fever virus antigen peptide P72 (SEQ ID NO: 198), Plasmodium antigen peptide CSP (SEQ ID NO: 199), and cytomegalovirus antigen peptide gB (SEQ ID NO: 200). Similarly, a PolyA was extended from the 5' end of the short chain at the designed position to form a capture strand. A C-terminal azide-modified SpyTag peptide glue (SpyTag-N3) and a 5'-terminal dibenzocyclooctene (DBCO)-modified PolyT (DBCO-PolyT) were covalently linked via click chemistry to form a SpyTag-PolyT conjugate. The SpyTag peptide was then engraved onto ICO via DNA complementary hybridization between the PolyT in the SpyT-PolyT conjugate and the protruding PolyA in the capture strand. Finally, the fusion proteins of SpyCatcher and different antigenic peptides were linked to ICO via spontaneous covalent isopeptide bond formation between SpyCatcher and SpyTag, ultimately achieving site-specific display of antigens on the surface of ICO.

[0149] To evaluate the in vivo immunogenicity of the ICO-HA influenza A antigen peptide nanovaccine, 6-week-old BALB / c mice were immunized intramuscularly twice with different ICO-HA nanovaccines containing 2 μg of HA (SEQ ID NO: 197) and formulated with AddaVax adjuvant ( FIG13 a ). Phosphate-buffered saline (PBS), HA monomer, ICO, and HA+ICO (physical mixture) were used as controls. By enzyme-linked immunosorbent assay (ELISA), the serum titers of HA-specific IgG in the HA and ICO-HA-6c×5-10 nm groups were 10 and 10, respectively, at week 2 after priming. 2 and 10 3 After boosting, the average HA-specific IgG antibody titer elicited by the ICO-HA-6c×5-10nm nanovaccine was higher than 10 5 , while HA monomer, HA+ICO and other ICO-HA nanovaccines including ICO-HA-1c×5-10nm, ICO-HA-1c×5-40nm and ICO-HA-6c×1-10nm only produced 10 3 -10 4 In conclusion, the ICO-HA-6c×5-10 nm nanovaccine is highly immunogenic compared to HA monomer.

[0150] At the same time, we constructed an ICO-CSP malaria antigen peptide nanovaccine using the same method and evaluated its in vivo immunogenicity. Six-week-old BALB / c mice were immunized intramuscularly twice with ICO-CSP nanovaccines containing 2 μg of CSP antigen (SEQ ID NO: 199) in different distributions and adjuvants (Figure 14a). Phosphate-buffered saline (PBS), CSP monomer, ICO, and CSP monomer + ICO (physical mixture) were used as controls. Enzyme-linked immunosorbent assay (ELISA) assays showed that at week 2 after priming, the serum titers of CSP-specific IgG in the CSP monomer and ICO-CSP-6c×5-10 nm groups reached 84.5 and 294.8, respectively (Figure 14b). After boosting, the average titer of CSP-specific IgG elicited by the ICO-CSP-6c×5-10 nm nanovaccine was higher than 10 4 The titers of CSP-specific IgG produced by CSP alone, CSP+ICO and other ICO-CSP nanovaccines including ICO-CSP-1c×5-10nm, ICO-CSP-1c×5-40nm and ICO-CSP-6c×1-10nm were all lower than 10 4 ( FIG14 c ) In conclusion, the ICO-CSP-6c×5-10 nm nanovaccine has a higher immunogenicity than CSP alone.

[0151] Similarly, to evaluate the immunogenicity of the ICO-P72 African swine fever recombinant antigen peptide nanovaccine in vivo, 6-week-old BALB / c mice were intramuscularly immunized twice with ICO-P72 nanovaccines containing 10 μg of P72 recombinant antigen protein (SEQ ID NO: 198) and formulated with AddaVax adjuvant ( FIG15 a ). Phosphate-buffered saline (PBS), P72 recombinant antigen protein, ICO, and P72 recombinant antigen protein + ICO (physical mixture) were used as controls, respectively. As detected by enzyme-linked immunosorbent assay (ELISA), the serum titers of HA-specific IgG in the P72 recombinant antigen protein and ICO-P72-6c×5-10nm groups reached 171.9 and 1814.6, respectively, at week 2 after priming immunization ( FIG15 b ). After boosting immunization, the P72-specific IgG titer elicited by the ICO-P72-6c×5-10nm nanovaccine was higher than 10 4 The titers of P72-specific IgG produced by P72 recombinant antigen protein alone, P72 recombinant antigen protein + ICO, and other ICO-P72 nanovaccines including ICO-P72-1c×5-10nm, ICO-P72-1c×5-40nm, and ICO-P72-6c×1-10nm were all lower than 10 4( Figure 15 c ) In conclusion, the ICO-P72-6c×5-10 nm nanovaccine has a higher immunogenicity than the P72 recombinant protein alone.

[0152] To evaluate the in vivo immunogenicity of the ICO-gB cytomegalovirus antigen peptide nanovaccine, 6-week-old BALB / c mice were intramuscularly immunized twice with different ICO-gB nanovaccines containing 5 μg of gB protein (SEQ ID NO: 200) and adjuvants ( FIG16 a). Phosphate-buffered saline (PBS), gB monomer, ICO, and HA+ICO (physical mixture) were used as controls. Enzyme-linked immunosorbent assay (ELISA) assay showed that the serum titers of gB-specific IgG in the HA and ICO-gB-6c×5-10 nm groups reached 108.7 and 946.1, respectively, at week 2 after priming ( FIG16 b). After boosting, the average titer of gB-specific IgG elicited by the ICO-gB-6c×5-10 nm nanovaccine was higher than 10 5 , while gB monomer, gB+ICO and other ICO-gB nanovaccines including ICO-gB-1c×5-10nm, ICO-gB-1c×5-40nm and ICO-gB-6c×1-10nm only produced 10 3 -10 4 In conclusion, the ICO-gB-6c×5-10 nm multivalent nanovaccine is highly immunogenic compared to the gB monomer.

[0153] Example 5: Construction of ICO-F and ICO-gE Nanovaccines and In Vivo Evaluation

[0154] Using a non-covalent attachment method, such as the reaction of NTA with His, the 5' end of the short chain at the designed position of ICO in Example 1 is modified with an amino group. After reaction with NTA-NHS, NTA is attached to the 5' end of the capture chain to form ICO-NTA. The antigen peptide is then arranged on the ICO by linking the NTA to the His tag on the antigen peptide.

[0155] To evaluate the in vivo immunogenicity of the ICO-F antigen peptide (SEQ ID NO: 195) nanovaccine against RSV virus, 6-week-old BALB / c mice were intramuscularly immunized twice with different ICO-F nanovaccines containing 10 μg of F protein and adjuvants ( FIG. 17 a ). Phosphate-buffered saline (PBS), F protein monomer, ICO, and F protein + ICO (physical mixture) were used as controls, respectively. As detected by enzyme-linked immunosorbent assay (ELISA), the serum titers of F protein-specific IgG in the F protein and ICO-F-6c×5-10 nm groups reached 200.9 and 5029.4, respectively, at week 2 after priming immunization ( FIG. 17 b ). After boosting immunization, the F protein-specific IgG titers elicited by the ICO-F-6c×5-10 nm nanovaccine were higher than 10 5 , while F protein monomer, F+ICO and other ICO-F nanovaccines including ICO-F-1c×5-10nm, ICO-HA-1c×5-40nm and ICO-F-6c×1-10nm only produced 10 3 -10 4 In conclusion, the ICO-F-6c×5-10 nm nanovaccine is highly immunogenic compared to the F protein monomer.

[0156] Similarly, we constructed a herpes zoster nanovaccine of ICO-gE (SEQ ID NO: 196) using a non-covalent linking method and evaluated its in vivo immunogenicity. Six-week-old BALB / c mice were intramuscularly immunized twice with different ICO-gE nanovaccines containing 5 μg of gE protein and adjuvants (Figure 18a). Phosphate-buffered saline (PBS), gE protein monomer, ICO, and gE protein + ICO (physical mixture) were used as controls. Enzyme-linked immunosorbent assay (ELISA) showed that the serum titers of gE protein-specific IgG in the F protein and ICO-gE-6c×5-10 nm groups reached 145.4 and 5159.3, respectively, at week 2 after priming (Figure 18b). After boosting, the gE-specific IgG titers elicited by the ICO-gE-6c×5-10 nm nanovaccine were higher than 8×10 4 , while gE protein monomer, gE+ICO and other ICO-gE nanovaccines including ICO-gE-1c×5-10nm, ICO-gE-1c×5-40nm and ICO-gE-6c×1-10nm only produced 10 3 -10 4 In conclusion, the ICO-gE-6c×5-10 nm nanovaccine is highly immunogenic compared to the gE protein monomer.

[0157] in conclusion

[0158] In summary, using DNA origami as a platform for the rational design and precise assembly of SARS-CoV-2 RBD nanovaccines, we constructed an icosahedral DNA origami nanostructure with a shape and size close to that of SARS-CoV-2. Our previously established "engraving printing" strategy was used to perform site-specific antigen display on icosahedral DNA origami to screen for surface antigen patterns that maximize B cell activation, including antigen spacing, intracluster antigen copy number, and cluster number. A nanovaccine with the optimal surface RBD pattern, ICO-RBD-6c × 5-10 nm, was selected as a candidate vaccine.

[0159] In vivo immune efficacy evaluation found that the optimized ICO-RBD nanovaccine can induce stronger short-term and long-term immune effects compared with free RBD and other models. Rationally designed molecular vaccines can induce strong B cell responses and improve the efficacy of SARS-CoV-2 vaccines. The design of the relationship between antigen configuration and immunogenicity used in this article can be extended to the study of other viral vaccines. Recently, chimeric vaccines have been developed that provide broad-spectrum immunity to multiple SARS-CoV-2 variants, and multivalent chimeric vaccine designs designed using DNA nanotechnology are also expected to improve immune protection against multiple variants. At the same time, in vivo immune evaluation found that compared with other free antigenic peptides, including RSV antigenic peptide F glycoprotein, herpes zoster antigenic peptide gE protein, influenza A antigenic peptide hemagglutinin HA, African swine fever virus antigenic peptide P72, Plasmodium antigenic peptide CSP and cytomegalovirus antigenic peptide gB, the optimized ICO-HA, ICO-P72, ICO-CSP, ICO-gB, ICO-F and ICO-gE nanovaccines can induce stronger in vivo neutralizing antibody titers. This type of multivalent vaccine design based on DNA nanotechnology is expected to become a highly effective antiviral vaccine carrier.

[0160] Statistical analysis

[0161] Data are expressed as mean ± standard deviation (SD). Multiple groups were compared using one-way analysis of variance (ANOVA) and Tukey's post hoc test. P < 0.05 was considered statistically significant.

[0162] Sequence Listing

[0163] SARS-CoV-2 receptor binding domain (RBD) (GenBank: WEU80698.1) (SEQ ID NO: 194)

[0164] F fusion protein (F) [Respiratory syncytial virus] (GeneID: 1494475) (SEQ ID NO: 195)

[0165] gE protein [human alphaherpesvirus 3] (GenBank: AAY57748) (SEQ ID NO: 196)

[0166] Hemagglutinin [Influenza A virus (H1N1)] (GenBank: ADC45736.1) (SEQ ID NO: 197)

[0167] African swine fever virus P72 (GenBank: QNC71679.1) (SEQ ID NO: 198)

[0168] Cyclosporine protein (CSP) [Plasmodium vivax] (GenBank: AAA29526.1) (SEQ ID NO: 199)

[0169] Glycoprotein B [Human beta herpesvirus 5] GenBank: ABQ23592.1 (SEQ ID NO: 200)

Claims

1. A subunit vaccine based on a DNA origami structure, comprising a DNA origami structure and an antigen peptide, wherein: The antigenic peptides are distributed in clusters on the DNA origami structure, with the number of antigenic peptides in each cluster being 2-6, preferably 5, and the distance between the antigenic peptides in each cluster and the nearest antigenic peptide is about 5-20 nm, preferably about 10 nm, and each DNA origami structure has 1-20, preferably at least 6 clusters of antigenic peptides.

2. The subunit vaccine of claim 1, wherein: - the DNA origami structure is a polyhedron, preferably a regular polyhedron such as a regular icosahedron, and / or - the diameter of each DNA origami structure is about 20-100 nm, preferably about 80-95 nm, and / or -The antigen peptide is linked to the surface of the DNA origami structure by covalent or non-covalent means.

3. The subunit vaccine of claim 1 or 2, wherein the antigenic peptide is an immunogen derived from a pathogen such as a virus, preferably, the pathogen is selected from influenza virus, coronavirus, respiratory syncytial virus, herpes zoster virus, African swine fever virus, Plasmodium and cytomegalovirus.

4. The subunit vaccine of any one of claims 1 to 3, wherein the antigenic peptide is selected from the group consisting of the S protein receptor binding region of SARS-CoV-2, respiratory syncytial virus fusion (F) glycoprotein, herpes zoster virus glycoprotein E protein, influenza virus such as influenza A (H1N1) hemagglutinin, African swine fever virus P72 protein, Plasmodium cyclosporin and cytomegalovirus glycoprotein B, Preferably, the antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 194-200, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence and having immunogenicity, More preferably, the antigenic peptide is the S protein receptor binding region of SARS-CoV-2, preferably having the sequence shown in SEQ ID NO: 194 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto and being immunogenic.

5. The subunit vaccine of any one of claims 1-4, wherein each DNA origami structure has at least 6 clusters, for example 6-12 clusters, wherein each cluster has 5 antigenic peptides and / or the distance between each antigenic peptide in each cluster and the nearest antigenic peptide is about 10 nm, more preferably, the 5 antigenic peptides in at least one cluster form the vertices of a pentagon, preferably a regular pentagon.

6. The subunit vaccine of any one of claims 1-5, wherein the antigen peptide is connected to the DNA origami structure through the interaction of a linker molecule and a matching linker molecule such as a Spycatcher and a Spytag peptide, preferably, the Spycatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 202 and / or the Spytag peptide comprises the amino acid sequence shown in SEQ ID NO:

201.

7. The subunit vaccine according to any one of claims 1 to 6, wherein the DNA origami structure is an icosahedron formed by the DNA sequence shown in SEQ ID NO: 1-193.

8. The subunit vaccine of any one of claims 1 to 7, wherein: - the subunit vaccine comprises a DNA origami structure formed by the short-chain DNA shown in SEQ ID NO: 1, 3-14, 16-28, 30-41, 43-51, 53-54, 56-57, 59-64, 66-70, 72-81, 83-87, 89-93, 96-101, 103-107, 109-113, 116-121, 123-127, 129-130, 132-142, 144-152, 154-158, 160-164, 167-180, 182, 184-185, 187-190, 193 and the capture DNA shown in SEQ ID NO: 204-233, and an adapter molecule-polyT such as Spytag-polyT connected thereto, and / or - A fusion protein formed by the antigenic peptide and a cooperating adaptor molecule such as a Spycatcher peptide, and / or - The fusion protein is linked to the DNA origami structure through the interaction of an adaptor molecule and a complementary adaptor molecule such as Spycatcher and Spytag peptides.

9. A method for preparing the subunit vaccine according to any one of claims 1 to 8, comprising: (a) providing a DNA origami structure, preferably having a diameter of about 20-100 nm, more preferably about 80-95 nm, (b) connecting the antigenic peptides to the DNA origami structures, respectively, wherein the antigenic peptides are distributed in clusters on the DNA origami structure, the number of antigenic peptides in each cluster is 2-6, preferably 5, and the distance between any antigenic peptide and the nearest antigenic peptide is about 5-20 nm, preferably about 10 nm, and the number of clusters per DNA origami structure is 1-20, preferably at least 6, and (c) Harvesting of the formed DNA origami-based subunit vaccine.

10. A pharmaceutical composition comprising the subunit vaccine according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

11. Use of the subunit vaccine according to any one of claims 1 to 8 in the preparation of a medicament for preventing or treating pathogen infection, preferably, the pathogen is selected from influenza virus, coronavirus, respiratory syncytial virus, herpes zoster virus, African swine fever virus, malarial parasite and cytomegalovirus.

Images