WO2022088953A1

WO2022088953A1 - Sars-cov-2 rbd conjugated nanoparticle vaccine

Info

Publication number: WO2022088953A1
Application number: PCT/CN2021/115957
Authority: WO
Inventors: 曾木圣; 康银峰; 孙聪; 冯国开
Original assignee: 中山大学; 中山大学肿瘤防治中心 (中山大学附属肿瘤医院、中山大学肿瘤研究所)
Priority date: 2020-10-29
Filing date: 2021-09-01
Publication date: 2022-05-05
Also published as: CN111991556B; CN111991556A

Abstract

The present invention provides a SARS-CoV-2 RBD conjugated nanoparticle vaccine, comprising: a) a nanoparticle carrier obtained by self-assembly of a carrier protein expressed in fusion with SpyCatcher; b) RBD antigen of SARS-CoV-2 virus expressed in fusion with SpyTag, the carrier protein being selected from mi3 and I53-50, and the carrier protein and the antigen being covalently linked by SpyCatcher-SpyTag.

Description

SARS-CoV-2 RBD Conjugated Nanoparticle Vaccine

technical field

The invention relates to the field of immunomedicine, in particular to a SARS-CoV-2 RBD conjugated nanoparticle vaccine.

Background technique

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a pathogen that causes acute lower respiratory tract infection with high lethality and can cause diseases such as novel coronavirus pneumonia (COVID-19). Due to the strong infectivity, rapid spread and high lethality of SARS-CoV-2, there is currently no effective specific medicine and vaccine for prevention and treatment, so the development of preventive vaccines against the virus is particularly urgent.

The S protein is a trimeric glycoprotein, a class I virus fusion protein, which also includes HIV glycoprotein 160 (Env), influenza hemagglutinin (HA), Paramyxovirus F and Ebola virus glycoprotein. The S protein can bind to the virus receptor of the host cell and is a key protein that determines the virus's invasion into susceptible cells. The role of the S protein in receptor binding and membrane fusion makes it an ideal target for vaccine and antiviral development, which can induce antibodies to block viral binding and fusion or neutralize viral infection. Among all the structural proteins of SARS-CoV-2, the S protein is the major antigenic component responsible for inducing host immune responses, neutralizing antibodies and protective immunity against coronavirus infection. The immunogenicity and protein yield of S protein limit the large-scale production of S protein, so subunit vaccines mainly focus on the receptor-binding domain (RBD) of S protein. However, although RBD-based vaccines have been extensively studied, the immunogenicity of RBD subunits remains low for various reasons, hindering the application of RBD subunits. To increase immunogenicity, scientists have worked to improve RBD.

So far, a variety of SARS-CoV-2 vaccines have been developed around the world, mainly including inactivated vaccines, subunit vaccines, viral vector vaccines and nucleic acid vaccines, and 7 vaccines have been approved for clinical phase III, but none have been launched to prevent There is still a need to develop a preventive vaccine against SARS-CoV-2 that produces high titers of neutralizing antibodies.

SUMMARY OF THE INVENTION

The present invention relates to an immunogenic complex comprising:

a) The nanoparticle carrier obtained by self-assembly of the carrier protein fused and expressed with SpyCatcher;

b) RBD antigen of SARS-CoV-2 virus expressed in fusion with SpyTag;

The carrier protein is selected from mi3 and I53-50;

The carrier protein and the antigen are covalently linked through SpyCatcher-SpyTag;

in:

The amino acid sequence of the RBD antigen is as shown in SEQ ID NO: 1;

The amino acid sequence of the mi3 is shown in 3; the I53-50 protein is assembled from the trimeric I53-50A1.1PT1 and the pentameric I53-50B.4PT1, and the I53-50A1.1PT1 contains SEQ ID NO : the amino acid sequence shown in 4; the I53-50B.4PT1 contains the amino acid sequence shown in SEQ ID NO: 5.

The present invention also relates to a nanoparticle vaccine comprising the immunogenic complex as described above.

According to a further aspect of the present invention, it also relates to a kit comprising a nanoparticle vaccine as described above, and a container for inoculating said nanoparticle vaccine.

The present invention also relates to the preparation method of the above-mentioned immunogenic complex, comprising:

The fusion protein in the a) component and the b) component is expressed, and the immunogenic complex obtained by co-incubating after purification, self-assembly.

The present invention also relates to the application of the above-mentioned immunogenic complex or the above-mentioned nanoparticle vaccine in the preparation of a medicament for the treatment of novel coronavirus pneumonia.

Compared with the prior art, the beneficial effects of the present invention are:

Compared with the monomeric RBD, the immunogenic complex obtained by the self-assembly of polypeptides with specific sequences in the present invention has significantly enhanced antigenic properties, and can induce higher titers of neutralizing antibodies, and the neutralizing antibodies hinder ACE2 and ACE2 and The ability of CB6 antibody to bind to RBD is stronger. After testing, animals do not produce obvious cellular immunity after immunization, and the vaccine is safer to use, easy to produce, and has high yield.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the accompanying drawings required in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is the structure and structural characteristics of RBD-coupled nanoparticles in an embodiment of the present invention;

(A) RBD nanoparticle design sketch; the flow chart on the left briefly introduces the use of the SpyTag-SpyCatcher system to transform RBD and nanoparticle backbone proteins; the particle in the figure shows that the ideally coupled nanoparticle is fully coupled to the outside of the RBD;

(B) Construction of target protein expression plasmids in different expression systems of E. coli and HEK293F cells;

(C) The results of SDS-PAGE of RBD monomer, RBD-conjugated nanoparticles and unconjugated unloaded nanoparticles under SDS-PAGE under reducing agent; as shown in the figure, the efficiency of covalent coupling is extremely high, and it can be seen that RBD monomers and unlinked nanoparticle scaffold proteins were not seen in the lanes of RBD-coupled nanoparticles;

(D) Distribution of RBD monomers, unconjugated nanoparticles and RBD-conjugated nanoparticles in size exclusion chromatography Superose 6 increase 10/300GL; after RBD-SpyTag was covalently coupled with △N1-SpyCatcher-NPs Peak shift can be observed;

(E) Dynamic light scattering (DLS) profiles of RBD monomer, RBD-coupled nanoparticles, and uncoupled nanoparticles; the diameter of the coupled nanoparticles can be seen to increase;

FIG. 2 is the verification of the assembly properties and physical characteristics of the assembled particles in one embodiment of the present invention;

The unconjugated nanoparticles and RBD-coupled nanoparticles observed under the negative staining electron microscope showed obvious burr-like protrusions on the outside of the RBD-coupled nanoparticles;

Fig. 3 is the antigenic identification of RBD monomer and RBD-conjugated nanoparticle in one embodiment of the present invention;

(A) The ability of RBD and RBD nanoparticles to bind ACE2 and CB6 antibodies was detected by ELISA, using two-way ANOVA to analyze the difference in binding titers between monomers and nanoparticles, and using Dunnett correction;

(B) BLI kinetics to detect the binding ability of RBD and RBD nanoparticles to ACE2;

(C) BLI kinetics to detect the binding ability of RBD and RBD nanoparticles to CB6;

Fig. 4 is the immunogenicity identification of RBD monomer and RBD-conjugated particle in one embodiment of the present invention;

(A) Flow chart of animal immunization;

(B) Serum antibody titers of immunized mice, shown in the figure according to different adjuvants, using two-way ANOVA to analyze the difference in antibody titers between monomers and nanoparticles, and using Dunnett correction, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001;

(C) BLI serum competition experiment, the sera of the immunized group using AddaVax as an adjuvant were subjected to a competition binding experiment with ACE2 or CB6 antibody, wherein Rc was the RBD competition binding signal of ACE2 and CB6 under the binding of different titers of serum, Ro is the RBD binding signal of ACE2 and CB6 without serum binding;

(D) Heat map of competition experiment, using (Ro-Rc)/R0 to indicate the degree of competition, and brighter colors show that the ability of serum to compete with ACE2 and CB6 for binding to RBD is stronger at different serum titers;

Fig. 5 is immune serum neutralization experiment in one embodiment of the present invention;

(A) SARS-CoV-2 pseudovirus neutralization experiment, the titer is expressed by NT ₉₀ , it can be seen that the serum neutralization ability of RBD nanoparticles is stronger than that of RBD monomer;

(B) SARS-CoV-2 live virus neutralization experiment, the titer is expressed by plaque reduction NT ₉₀ (FRNT ₉₀ ), it can also be seen that the serum neutralization ability of RBD nanoparticles is stronger than that of RBD monomer, and the titers of different groups are compared. Statistical tests were performed with an unpaired two-tailed Mann-Whitney U test, where *p<0.05;**p<0.01;***p<0.001;****p<0.0001;

6 is the immune cell characteristics of the draining lymph nodes of the mice after each immunization in an embodiment of the present invention;

(A) The germinal center B cells were identified by flow cytometry using B220, IgD-low, GL7 and CD95 as markers. The GC B cells in the lymph nodes of the mice in each group were counted and found that there was no significant difference between the groups. difference;

(B) Tfh follicular helper T cells were identified by flow cytometry using CD4, CD44, PD-1 and CXCR5 positivity as markers, and no significant difference was found between the groups after statistics;

(C) Cytokine-releasing CD4+ cells were identified by co-expressing IFN-γ, IL2 or TNF-α as markers, and no significant differences were found between the groups;

(D) CD8+ T cells released by cytokines were identified by co-expressing IFN-γ, IL2 or TNF-α as markers, and no significant difference was found between the groups;

Figure 7 shows the immune cell characteristics in the spleen of mice after each immunization;

(A) Cytokine-releasing CD4+ (A) cells were identified by co-expressing IFN-γ, IL2 or TNF-α as markers, and no significant difference was found between the groups;

(B) Cytokine-releasing CD8+ T (B) cells were identified by co-expressing IFN-γ, IL2 or TNF-α as markers, and no significant differences were found between the groups.

Detailed ways

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of illustration and not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

Therefore, it is intended that this invention covers such modifications and changes as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or will be apparent from the following detailed description. It should be understood by those of ordinary skill in the art that this discussion is a description of exemplary embodiments only, and is not intended to limit the broader aspects of the invention.

The present invention relates to an immunogenic complex comprising:

b) RBD antigen of SARS-CoV-2 virus expressed in fusion with SpyTag;

The carrier protein is selected from Ferritin, mi3 and I53-50;

in:

The amino acid sequence of the RBD antigen is as shown in SEQ ID NO: 1;

The amino acid sequences of the Ferritin and the mi3 are shown in SEQ ID NOs: 2 and 3 in turn; the I53-50 protein is assembled from the trimeric I53-50A1.1PT1 and the pentameric I53-50B.4PT1, The I53-50A1.1PT1 contains the amino acid sequence shown in SEQ ID NO:4; the I53-50B.4PT1 contains the amino acid sequence shown in SEQ ID NO:5.

The Ferritin protein is derived from the hybrid ferritin of bullfrog and Helicobacter pylori-bullfrog hybrid, which is an octahedron composed of 24 subunits. The Ferritin protein has N8Q and N19Q point mutations at residue 8 of the bullfrog ferritin portion and residue 19 of the H. pylori ferritin portion to avoid potential glycosylation sites; and residues of the H. pylori ferritin 7(I7E) was point mutated to preserve the salt bridge.

The carrier protein mi3 protein was point mutated by KDPG aldolase C76A and C100A to avoid potential disulfide-mediated heterogeneity. It is an icosahedron consisting of 60 subunits.

Carrier protein I53-50 is a 20-mer I53-50A1.1PT1 trimeric and 12 pentameric I53-50B.4PT1 20-mer assembled. Preferably, 20 trimers I53-50A1.1PT1 and 12 pentamers I53-50B.4PT1 are assembled in vitro with a molar mass ratio of 1:1-3.

In certain embodiments of the present invention, the SpyCatcher in component a) is fused to the carrier protein via a linker peptide.

In certain embodiments of the invention, the SpyTag in component b) is fused to the RBD antigen via a linker peptide.

In some embodiments, the number of amino acids of the linking peptide is 1-30; , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

In some embodiments, the amino acids of the linking peptide are nonsense polypeptides that do not have additional functions other than linking (eg, protein localization, enzyme cleavage site, etc.).

In some embodiments, the linker peptide is a flexible linker peptide.

In some embodiments, the amino acid sequence of the linking peptide is selected from one or more of Gly, Ser, Pro, Ala, and Glu.

In some embodiments, the amino acid sequence of the linking peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.

Wherein (GGS)n means that there are n GGS repeats, for example (GGS) ₄ means GGSGGSGGSGGS, and the same is true for others.

In some embodiments, the amino acid sequence of the linking peptide in component a) is from (GGS) ₄ .

In some embodiments, the amino acid sequence of the linking peptide in component b) is GSGGSGGSG.

In some embodiments, the SpyCatcher is N-terminal to the carrier protein.

In some embodiments, the SpyTag is C-terminal to the carrier protein.

In some embodiments, the SpyTag contains the amino acid sequence set forth in SEQ ID NO:6.

In some embodiments, the SpyCatcher contains the amino acid sequence set forth in SEQ ID NO:7.

In some embodiments, component b) is ΔN1-SpyCatcher-Ferritin, which contains the amino acid sequence set forth in SEQ ID NO: 9; or

b) The component is ΔN1-SpyCatcher-mi3, which contains the amino acid sequence shown in SEQ ID NO: 10; or

b) The component is ΔN1-SpyCatcher-I53-50, which contains the I53-50 protein assembled from the trimeric ΔN1-SpyCatcher-I53-50A1.1PT1 and the pentameric I53-50B.4PT1, wherein ΔN1 - SpyCatcher-I53-50A1.1PT1 contains the amino acid sequence shown in SEQ ID NO:11, and I53-50B.4PT1 contains the amino acid sequence shown in SEQ ID NO:5.

In some embodiments, pharmaceutically acceptable carriers and/or adjuvants are also included.

Examples of pharmaceutically acceptable carrier ingredients include binders (syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), fillers (lactose, sucrose, starch, calcium phosphate, sorbitol, etc.) , glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, etc.), disintegrating agents (starch, microcrystalline cellulose, etc.), wetting agents (sodium lauryl sulfate (sodium lauryl sulfate) lauryl sulphate, etc.), suspending agents (sorbitol, syrup, methylcellulose, glucose syrup, gelatin, hydrogenated edible fats, etc.), emulsifiers (lecithin, sorbitol monooleate, gum arabic) etc.), non-aqueous carriers (almond oil, fractionated coconut oil or hydrophobic esters such as glycerol, propylene glycol, ethanol, etc.), preservatives (methyl paraben or propyl paraben, sorbic acid, etc.), fragrances ( Synthetic flavors, natural flavors, etc.), sweeteners (sucrose, stevia, xylitol, etc.), pH adjusters (sodium bicarbonate, potassium carbonate, etc.), powders (pigments, dyes, resins, etc.), thickeners ( gum arabic, methylcellulose, etc.), antioxidants (vitamin C, vitamin E, etc.), etc.

The vaccine provided by the present invention preferably further comprises an adjuvant. Adjuvants suitable for use in the vaccines of the present invention include adjuvants that enhance antibody responses to B cell epitopes in the recombinant influenza virus, and adjuvants that enhance cell-mediated responses to T cell epitopes in the recombinant influenza virus. adjuvant. These adjuvants are well known in the art.

In some embodiments, the adjuvant is selected from the group consisting of Sigma Adjuvant Systerm, AddaVax, Squalene, Muramyl Dipeptide, MF59, AS03, Monophosphatidyl Lipid A, Flagellin, CpG-ODN, Poly(I: C), and one or more of the small molecules of aluminum or calcium salts. These adjuvants are well known in the art and are available through several commercial sources. Among them, the preferred adjuvants are Sigma Adjuvant Systerm and/or AddaVax.

In some embodiments, the vaccine is a water-in-oil emulsion having an aqueous phase and an oily phase.

In some embodiments, the vaccine is an oil-in-water emulsion having an aqueous phase and an oily phase.

Vaccines are typically formulated for parenteral administration. Vaccination is typically by nasal route, but oral and subcutaneous (SC), intramuscular (IM), intravenous (IV), intraperitoneal (IP) or intradermal (ID) injections are also contemplated by the present invention. .

The vaccines described above are administered in a manner compatible with the dosage formulation, and in amounts such as therapeutically effective and immunogenic effective amounts. The amount administered depends on the subject being treated, the ability of the subject's immune system to synthesize antibodies, and the degree of protection expected. The exact amount of active ingredient to be administered will depend on the judgment of the physician and will vary from individual to individual. Appropriate schedules of initial administration and booster vaccinations may also vary, but typically an additional injection or other administration occurs at some interval (weeks or months) after the first administration.

Another embodiment of the present invention relates to a kit comprising a nanoparticle vaccine as described above, and a container for administering the nanoparticle vaccine.

The inoculation container is preferably a medical syringe.

According to yet another aspect of the present invention, it also relates to the use of the above-mentioned immunogenic complex or the above-mentioned nanoparticle vaccine in the preparation of a medicament for the treatment of novel coronavirus pneumonia.

The present invention further provides a method of protecting a subject from infection by a SARS-CoV-2 virus, comprising administering to said animal an effective amount of a nanoparticle vaccine according to the present invention.

The subjects for the above purposes can refer to patients or animals suspected of carrying SARS-CoV-2, especially mammals, such as bats and civet cats; preferably primates, more preferably humans.

In some embodiments, subjects include infected individuals, recovered individuals, asymptomatic infected individuals, vaccinated individuals, and the like.

An effective amount is defined as the amount of the vaccine that will induce an immune response in the individual to which it is administered, resulting in the development of a secretory, cellular and/or antibody-mediated immune response in the individual against the vaccine. Said secretory, cellular and/or antibody-mediated immune response to the vaccine is also effective against challenge with virulent influenza strains.

The effective amount is preferably administered orally or oronasally or intramuscularly.

In some embodiments, one or more administrations are administered.

In certain embodiments of the invention,

Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Unless otherwise stated, immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, etc. employed in the present invention are routine skills in the art. See Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (edited by F.M. Ausubel et al., (1987)); METHODS IN ENZYMOLOGY series (academic publishing company): " PCR2: A PRACTICAL APPROACH (edited by M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), Harlow and Lane, editor (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney editor (1987)) .

Example 1. Design of SARS-CoV-2 RBD Nanoparticle Vaccines

This example describes the design of Ferritin (24-mer), mi3 (60-mer) and I53-50 (120-mer) nanoparticles based on the SARS-CoV-2 RBD protein.

Considering that different nanoparticles can display different copy numbers of antigens on their surfaces, three SARS-CoV-2 RBD-conjugated nanoparticles were designed in this example: RBD-Ferritin, RBD-mi3 and RBD-I53-50.

Ferritin (SEQ ID NO: 2) is an octahedron consisting of 24 subunits. Ferritin protein is the lower subunit of bullfrog (Rana catesbeiana) ferritin (UniProt: P07797) N-terminal residues 2-9 fused to residues 3-167 of Helicobacter pylori non-heme ferritin by molecular biological means. In order to eliminate the potential influence of glycosylation site at the N-terminal, the present invention adopts N8Q and N19Q point mutations at residues 8 and 19 of bullfrog ferritin. To preserve the salt bridge between residues 6R and 14E of H. pylori-bullfrog Ferritin, we similarly created a point mutation at residue 7 (I7E) of H. pylori ferritin.

mi3 (SEQ ID NO:3) is an icosahedron composed of 60 subunits, derived from KDPG aldolase and based on the computer-designed and optimized I3-01 nanoparticle protein mutated C76A and C100A to avoid potential Disulfide bond-mediated heterogeneity.

I53-50 was assembled in vitro from 20 trimeric I53-50A1.1PT1 (SEQ ID NO:4) and 12 pentameric I53-50B.4PT1 (SEQ ID NO:5) in a molar mass ratio of 1:1 20-mer formed.

SARS-Cov-2 S protein, especially the Receptor Bingding domain (RBD), is a key protein that can bind to virus receptors in host cells, and is a key protein that determines virus invasion into susceptible cells. Its role in membrane fusion makes it an ideal target for vaccine and antiviral development. The SARS-CoV-2 virus (Wuhan-Hu-1, GenBank: MN908947) RBD gene (residues: 319-541) was optimized and synthesized by mammalian codon bias. In order to construct the RBD-SpyTag fusion protein, a 13-residue SpyTag (SEQ ID NO:6) was fused to the C-terminus of the RBD gene (SEQ ID NO:1) through a Gly-Ser linker to obtain the SEQ ID NO:8 sequence. In order to facilitate the purification and removal of tagged proteins, the HRV 3C site and the His tag of 6 histidines were fused to the C-terminus of the SpyTag gene. There is no SpyTag plasmid; in order to facilitate the purification of the target protein using the eukaryotic expression system, we fused a signal peptide to the N-terminus of the target gene, so that the target protein can be secreted into the supernatant, and finally SARS-CoV-2 S RBD-SpyTag (SEQ ID NO: 12). In order to construct Ferritin, mi3 and I53-50 conjugated to RBD, the ΔN1-SpyCatcher (SEQ ID NO: 7) gene was fused to the N-terminus of Ferritin, mi3 and I53-50.1PT1 gene through a (G2S) ₄ linker, respectively, and At the N-terminus of Ferritin and mi3, and the C-terminus of I53-50.1PT1, a His-tag of 6 histidines and HRV 3C site were fused to construct a carrier protein △N1-SpyCatcher-Ferritin (SEQ ID NO: 13), △N1 - SpyCatcher-mi3 (SEQ ID NO: 14) and ΔN1-SpyCatcher-I53-50.1PT1 (SEQ ID NO: 15). I53-50B.4PT1 (SEQ ID NO: 16) was designed according to published literature [Bale, JB, et al. Accurate design of megadalton-scale two-component icosahedral protein complexes. Science 353, 389-394 (2016)]. The protein genes expressed in prokaryotic cells are optimized and synthesized based on the codon preference of the OptimumGeneTM E. coli expression system of Nanjing GenScript Biotechnology Co., Ltd.

Example 2. Expression and purification of SARS-CoV-2 RBD nanoparticle-based vaccine

1. Experimental materials

(1) Vectors and strains required for constructing recombinant vectors: mammalian expression vector VRC8400, E. coli expression vector modified PET-28a+, E. coli competent DH5a cells, Rosseta cells.

Protein expression cell line: HEK293-F cells (derived from human embryonic kidney epithelial cells).

(2) Reagents and consumables: PCR enzyme and recombinase (purchased from Novozan Co., Ltd.), endonuclease (purchased from NEB), cell transfection reagent PEI-MAX (Polysciences, Inc., Cat. No. 24765- 1), the mammalian cell culture medium Union 293 medium (purchased from Shanghai Yonglian Biotechnology), histidine-tagged protein purification agarose magnetic beads (purchased from GE company), and other conventional reagents and consumables are commercialized.

(3) Genes: △N1-SpyCatcher-Ferritin (SEQ ID NO:18), △N1-SpyCatcher-mi3 (SEQ ID NO:19), △N1-SpyCatcher-I53-50A1.1PT1 (SEQ ID NO:20) , I53-50B.4PT1 (SEQ ID NO: 21). Both hACE2-8*his (residues: 19-615) and hACE2-hFc genes were optimized and synthesized by Nanjing GenScript Bio-Co., Ltd. OptimumGeneTM codon preference platform.

2. Steps

(1) The S protein receptor binding region gene (SEQ ID NO: 17) and hACE2-8*his and hACE2-hFc genes of SARS-CoV-2 virus were connected to mammalian cells by PCR amplification and restriction enzyme recombination methods, respectively. Expression vector VRC8400. Genes ΔN1-SpyCatcher-Ferritin (SEQ ID NO:18), ΔN1-SpyCatcher-mi3 (SEQ ID NO:19), ΔN1-SpyCatcher-I53-50A1.1PT1 (SEQ ID NO:20), I53-50B .4PT1 (SEQ ID NO: 21) was ligated into the modified prokaryotic expression vector pET-28a by PCR amplification and restriction enzyme recombination method.

(2) For eukaryotic expression system expression and purification, the method is as follows: VRC8400-RBD-SpyTag-8*His, VRC8400-RBD-8*His, VRC8400-hACE2-8*his and VRC8400-hACE2- The bacterial solution of hFc was inoculated into 1 L of TB medium at a volume ratio of 1:100, and cultured overnight at 37°C and 220 rpm. After the bacterial solution was centrifuged at 4500 rpm for 10 min, the bacterial cells were collected, resuspended, lysed and neutralized, and the recombinant plasmid of E. coli was extracted through steps such as ion exchange. HEK293F cells with a density of 1.0×10 ⁶ were transfected with plasmid: PEI-MAX=1:5, 1 mg/L, 5 days after transfection, and the cell supernatant was collected. After centrifugation, the supernatant was filtered through a 0.22 μm filter. For proteins carrying His tags, the supernatant flows through a Ni-NTA affinity chromatography column, imidazole is used to elute the target protein, concentrated and further purified by a gel chromatography column. The protein was concentrated, the concentration of purified protein was determined by BCA method, aliquoted, snap frozen in liquid nitrogen and stored at -80°C. For Fc-tagged proteins, the supernatant flows through a Protein A chromatographic column, washed with PBS for 10 column volumes, 0.2M glycine, pH 3.0, to elute the target protein, and the further purification method is the same as the His-tagged protein.

(3) For the prokaryotic expression system to express and purify the protein, the method is as follows: the recombinant vectors pET-28a-N1-SpyCatcher-Ferritin, pET-28a-△N1-SpyCatcher-mi3, pET-28a-△N1-SpyCatcher-I53- 50A1.1PT1, pET-28a-I53-50B.4PT1 were transformed into Escherichia coli competent Rosseta cells, positive clones were screened for resistance (kanamycin and chloramphenicol) and the bacteria of interest were expanded at 37°C. ℃ Add the chemical inducer isopropylthiogalactoside (IPTG) at a final concentration of 0.5mM to induce the expression of the target protein. After 16-20h induction, the cells were collected, crushed by high pressure, centrifuged to take the supernatant and filtered at 0.22 μm. The protein was purified by protein affinity chromatography and molecular sieve as above to obtain a high-purity target protein, that is, a recombinant nanoparticle protein. The purified △N1-SpyCatcher-I53-50A1.1PT1 and I53-50B.4PT1 were assembled in vitro at room temperature with a subunit ratio of 1:1, and the target protein was separated by a molecular sieve Superose 6 Increase 10/300 GL gel filtration column.

(4) Incubate the purified three nanoparticle proteins (>20μM) and the RBD-SpyTag protein of SARS-CoV-2 at room temperature with a subunit molar mass ratio of 8:1 or above for 12h, and the incubation buffer is: 50mM HEPES, pH 7.3, 300mM NaCl, after incubation, the conjugated mixture of nanoparticles and SARS-CoV-2 RBD-SpyTag protein was passed through Superose 6 Increase 10/300 GL gel filtration column to remove RBD monomer protein, and purified to obtain target protein.

3. Results

As shown in Figure 1C, high-purity RBD protein and three nanoparticles △N1-SpyCatcher-Ferritin, △N1-SpyCatcher-mi3, △N1-SpyCatcher-I53-50, and three nanoparticles were expressed and purified, respectively. RBD-Ferritin, RBD-mi3 and RBD-I53-50 nanoparticle vaccines conjugated with RBD proteins via the amide bond of SpyTag and ΔN1-SpyCatcher.

Example 3 Characterization of RBD-conjugated Nanoparticle Proteins

1. Experimental materials

(1) Reagents and consumables: 300 mesh copper mesh, disposable solvent-resistant micro-test tubes, etc. are commonly used commercial reagents and consumables.

(2) Instruments: 120KV transmission electron microscope (FEI, USA), Nano differential scanning fluorimetry (NanoDSF) Systems (NanoTemper Technologies) and Zetasizer Ultra (Malvern, UK).

2. Experimental steps

2.1 Negative staining TEM

(1) Dilute the RBD-conjugated nanoparticle protein and △N1-SpyCatcher-NPs (unconjugated empty particles) to a protein concentration of 0.05-0.2 mg/mL, pipette 10 μL of the protein onto a clean plastic film, and place the The carbon-coated copper mesh after discharge was placed in the protein solution for 2 min, gently blotted dry with filter paper, then washed twice with double distilled water, blotted with filter paper, and then incubated with 2% uranyl acetate for 2 min for staining, and Air dry naturally.

(2) The stained protein samples were subjected to transmission electron microscopy to observe the size and morphology of the particles.

2.2 Detection of particle size by Zetasizer Ultra

(1) Centrifuge the RBD-conjugated nanoparticle protein and ΔN1-SpyCatche-NPs protein for 10 min at 12,000 rpm, dilute the protein concentration with PBS to 0.5 mg/mL, add 40 μL of the sample to a disposable solvent-resistant micro-tube sample pool, statically Set 3min.

(2) Use the Zetasizer Ultra instrument of Malvern Company to detect the particle size of the nanoparticles, set the measurement angle to 173°, and determine the size distribution of the purified protein by measuring the intensity of scattered light.

2.3 Detection of Tm value and Tagg value of particle vaccine using NanoDSF Systems

(1) Centrifuge the RBD-conjugated nanoparticle protein and ΔN1-SpyCatche-NPs protein for 10 min at 12,000 rpm, dilute the protein concentration with PBS to a concentration of 0.5 mg/mL, add 10 μL of the sample to a quartz capillary, and put it into the card slot. Three replicates were set up for each sample.

(2) The parameters are set in the PR.ThermControl software, the heating rate is 1°C/min, and the heating range is 20°C-95°C.

(3) After Dsicovery Scan, confirm that the sample is correct, set 30% Excitation Power, and start Melting Scan.

3. Experimental results

As shown in Fig. 2, the transmission electron microscope images of RBD-conjugated nanoparticle proteins and △N1-SpyCatche-NPs proteins, the results show that all recombinant nanoparticle proteins formed uniform particles, and the three types of RBD-conjugated nanoparticles The particle diameter of the protein was slightly larger than that of the ΔN1-SpyCatche-NPs protein. Table 1 shows the size and distribution of the hydrated particle size of the RBD-conjugated nanoparticle protein and the ΔN1-SpyCatche-NPs protein.

The dynamic light scattering results show that, as shown in Table 1, the hydration radius of the recombinant nanoparticle protein is slightly increased compared with the results of transmission electron microscopy due to a layer of water on the outside of the recombinant nanoparticle protein. The RBD monomer protein is 8.98±0.03nm, △N1-SpyCatche-Ferritin is 28.75±0.18nm, RBD-Ferritin is 32.99±0.04nm, △N1-SpyCatche-mi3 is 41.87±0.39nm, RBD-mi3 is 55.19±0.49nm, △N1-SpyCatche-I53-50 was 46.54 ± 0.40 nm, and RBD-I53-50 was 50.67 ± 0.11 nm. Compared with monomeric nanoparticles, the RBD-conjugated nanoparticle protein was larger, indicating that the RBD protein was displayed on the outside of the ΔN1-SpyCatche-NPs protein. High-throughput protein stability analyzer analysis showed that the Tm values of RBD and RBD-conjugated nanoparticle proteins were around 45°C, whereas RBD-I53-50 aggregated at around 70°C (Table 1).

Table 1 Table of DLS and DSF results for nanoparticles

R _d : hydrodynamic diameter;

PDI: Dispersion index, when it is less than 0.2, it means that the particle distribution is single;

T _m1 : the first melting temperature;

T _m2 : the second melting temperature;

T _aggr : aggregation temperature;

The above parameters are all derived from the parameter values generated by the software.

Example 4. Antigenic properties of RBD-conjugated nanoparticle proteins

1. Experimental materials

(1) Reagents and consumables: protein A, protein G, protein A chip, SA chip, ELISA plate and EL-TMB color development kit are all commonly used commercial reagents and consumables.

(2) Antibodies: Goat anti-human, goat anti-mouse and goat anti-rabbit HRP-conjugated IgG H&L are commercial antibodies (Promega), and goat anti-rabbit hACE2 polyclonal antibodies (Yiqiao Shenzhou).

2. Experimental steps

2.1 Purification of CB6 antibody

(1) The heavy chain and light chain IgG genes of CB6 antibody were cloned into VH and VK expression vectors, the plasmids were extracted, and transfected into HEK293F cells at a DNA concentration of 1:1.2, and were separated and purified by Protein A matrix and gel column , to obtain CB6 antibody.

2.2 ELISA

2.2.1 Affinity of RBD-conjugated nanoparticles and the receptor hACE2 of SARS-CoV-2 virus

(1) Purification of hACE2-8*his protein. As shown in Example 2, hACE2 protein was expressed and purified in HEK293F cells.

(2) The receptor binding region (RBD-SpyTag) of SARS-CoV-2 virus S protein and RBD-conjugated nanoparticle proteins (RBD-Ferritin, RBD-mi3 and RBD-I53-50) and the control group BSA were added to PBS was used as a diluent, diluted to 1 μg/mL, coated with ELISA plates, 100 ng/well, with a total volume of 100 μL, coated overnight at 4°C, washed three times with PBST, and added with blocking solution (PBS containing 2% gelatin, 5% casino and 0.1% proclin 30) overnight blocking. Three replicates were set up for each sample.

(3) Dilute hACE2 protein. The initial protein concentration was 50 μg/mL, serially diluted 5-fold, with a total of 12 gradients, and the protein and protein complex were incubated at 37 °C for 2 h.

(4) Wash three times with PBST, add 1:5000 diluted polyclonal rabbit antibody against ACE2 protein, and incubate at 37°C for 1 h.

(5) Wash 5 times with PBST, add horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Promega Cat#W4011, RRID:AB_430833) diluted 1:5000, and incubate at 37°C for 45min.

(6) Wash 5 times with PBST to remove unbound antibodies, add TMB chromogenic solution, add stop solution after incubation for 15 min, read the absorbance values of OD ₄₅₀ and OD ₆₃₀ , and draw antigen and receptor affinity in GraphPad Prism 8 software curve.

2.2.2 Affinity of RBD-conjugated nanoparticles and RBD-specific antibody CB6

(1) The receptor binding region (RBD-SpyTag) of SARS-CoV-2 virus S protein and RBD-conjugated nanoparticle proteins (RBD-Ferritin, RBD-mi3 and RBD-I53-50) and the control group BSA were added to PBS was used as a diluent, diluted to 1 μg/mL, coated on ELISA plates, 100 ng/well, with a total volume of 100 μL, coated overnight at 4°C, washed three times with PBST, and added with blocking solution (PBS solution containing 2% gelatin, 5% casino and 0.1% proclin 30) overnight blocking. Three replicates were set up for each sample.

(2) Dilute the CB6 antibody. The initial antibody concentration was 250 μg/mL, serially diluted 5-fold, with a total of 12 gradients, and the protein and antibody complexes were incubated at 37 °C for 2 h.

(3) Wash 5 times with PBST, add 1:5000 diluted HRP-conjugated goat anti-human IgG secondary antibody (Promega, Cat#W4031, RRID:AB_430835), and incubate at 37°C for 45min.

(4) Wash 5 times with PBST, add TMB chromogenic solution, add stop solution after incubation for 15 min, read the absorbance values of OD ₄₅₀ and OD ₆₃₀ , and draw antigen and antibody affinity curves in GraphPad Prism 8 software.

2.3 Biofilm Interferometry (BLI) Analysis

BLI analysis was performed on an Octet Red 96 (Fortebio) instrument at 30°C and shaking at 1000 revolutions per minute. Signals are acquired at the default standard frequency (5.0Hz).

Kinetic assays: First, streptavidin (SA) biosensors (Fortebio) were pre-incubated in PBS (ThermoFisher) containing 0.05% Tween 20 (Sigma-Aldrich) for 15 min, the buffer was used throughout. To link the RBD protein to the biosensor, the RBD/ACE2/CB6 antibody was biotinylated using the EZ-link-Sulfo-NHS-biotin biotinylation kit (ThermoFisher) as follows.

Step 1: Calculate the amount of biotinylation reagent

1. Calculate the millimolar amount of the biotin reagent added to the reaction to be a 20-fold molar excess;

2. Calculate the volume of 10 mM biotin reagent solution added to the reaction;

Step 2: Add the calculated amount of 10 mM biotin reagent to the RBD/ACE2/CB6 protein, and incubate with PBS (5 mg/mL, 200 μL) for 30 minutes at room temperature.

Step 3: Desalting

Desalting column PD-10 (GE Pharmacia) was equilibrated with 10 mL of PBS, after equilibration, the reaction solution was added to the column, washed and eluted with 400 μL of PBS, respectively.

For kinetic assays, ACE/CB6-biotinylated protein diluted in buffer was captured on SA sensors at 2 μg/mL for 120 s after baseline binding for 60 s. RBD monomer or RBD-conjugated nanoparticles at equimolar mass concentrations of the same RBD monomer were then subjected to serial 2-fold serial dilutions, bound on the biosensor for 180 s, followed by dissociation for 300 s in 10 mM glycine pH 1.5 3 rounds of regeneration. Curve data were analyzed using ForteBio data analysis software. The original curve was adjusted to the baseline signal before performing a 1:1 binding model fit. All binding dissociation curves were then fitted globally and overall kinetic parameters (kD, kon, kdis, etc.) were plotted.

3. Results

To verify the antigenicity of three RBD-conjugated nanoparticles, we used SARS-CoV-2 ACE2 and antibody CB6 to verify the antigenicity of purified RBD-conjugated nanoparticles. ACE2 recognizes the receptor-binding region of the SARS-CoV-2 S protein. CB6 antibody is an antibody obtained from B cell sorting of recovered patients from COVID-19, which can recognize the receptor binding region of SARS-CoV-2 S protein and has the ability to neutralize SARS-CoV-2 virus. The results of this study are shown in Fig. 3A, ACE2 proteins can recognize both RBD-conjugated nanoparticles and RBD monomeric proteins, and there is no significant difference. Although CB6 antibodies can both recognize RBD-conjugated nanoparticles and RBD monomer proteins, the three RBD-conjugated nanoparticles can bind CB6 antibodies more strongly than RBD monomers, suggesting that RBD-conjugated nanoparticles Possibly more antigenic.

We applied biofilm layer interferometry to further examine the binding kinetics of RBD-conjugated nanoparticles. As shown in Figure 3B and Table 2, the RBD monomer and two RBD-conjugated nanoparticles, RBD-Ferritin and RBD-I53-50, were measured to bind the hACE2 receptor with an affinity constant value (kD) of 4.34E, respectively -09, 1.74E-08 and 1.00E-09M. However, RBD-mi3 nanoparticles dissociated very slowly, and the binding dissociation constant kD value between hACE2 and hACE2 reached 1.0e-12M, indicating that RBD-mi3 NPs performed better than RBD-Ferritin NPs and RBD-I53-50 NPs. higher antigenicity. The binding kinetics of RBD-conjugated nanoparticles to CB6 antibody were also determined. The binding ability of the three RBD-conjugated nanoparticles to the CB6 antibody was significantly stronger than that of the RBD monomer (Fig. 3C and Table 2), indicating that the three RBD-conjugated nanoparticles may have a specific BCR targeting SARS-CoV-2 RBD. higher affinity.

Table 2 Dynamic parameter table of BLI

k _D : binding constant;

k _on : association rate;

k _dis : dissociation rate.

Example 5 Immunogenicity of RBD-conjugated nanoparticles to BALB/c mice

1. Experimental materials

(1) Mice: female BALB/c mice of 6-8 weeks old.

(2) Adjuvants: commercial Sigma Adjuvant Systerm (SAS, Sigma) and AddaVax adjuvant (InvivoGen).

(3) Other reagent consumables are commercial conventional reagent consumables.

2. Experimental steps

Forty-five 6-8-week-old BALB/c female mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and randomly divided into 9 groups. The purified immunogen was diluted with PBS before immunization, mixed gently with an equal volume of AddaVax adjuvant (InvivoGen) or Sigma Adjuvant System (SAS, Sigma), and incubated at 4°C overnight at 40 RPM, so that the adjuvant and antigen were mixed at 4°C. Adhesive absorption at ℃. Mice in each group were immunized three times by subcutaneous injection at 0, 2, and 4 weeks. The immunization dose was 5 μg/unit of RBD monomer, and three RBD-conjugated NPs with the same molar mass as RBD monomer: RBD-mi3 (9.51 μg/unit), RBD-Ferritin (9.34 μg/unit) and RBD-I53-50 (11.91μg/only). PBS served as a negative control. Orbital blood was collected from mice 10 days after each immunization and placed at 37°C for 30 minutes to achieve adequate coagulation. Blood samples were taken, centrifuged at 12000 RPM at 4°C for 10 min, the upper serum was gently extracted, heat inactivated at 56°C for 30 min to inactivate complement factors and pathogens, and stored at -20°C for later use.

Indirect enzyme-linked immunosorbent assay: Serum from isolated samples was tested for total IgG titers, IgG1 and IgG2a antibody titers bound to anti-SARS-CoV-2 RBD in mouse serum by indirect enzyme-linked immunosorbent assay. The C-terminus of RBD diluted with PBS does not carry SpyTag monomer protein, the concentration is 1 μg/mL, coated with 96-well high-binding ELISA plate, 100 μL/well, and placed at 4°C overnight; configure the blocking solution as above, add the blocking solution to the ELISA plate , 100μL/well, overnight at 4°C; dilute serum: the initial serum concentration is 1:50, then use a 5-fold gradient, PBS as the diluent, serially dilute to 10 ^-8 , add it to the ELISA plate, incubate at 37°C for 1h ; Wash 5 times with PBST, add horseradish peroxidase-conjugated total goat anti-mouse IgG, IgG1 or IgG2a antibody at 1:5000 dilution respectively, incubate at 37°C for 45 min; Wash 5 times with PBST, add color developing solution TMB, The reaction was stopped by the addition of 2M _H2SO4 after ₁₅ min incubation at 37°C; absorbance was measured immediately at 450 nm and 630 nm with a SpectraMax Plus plate reader (Molecular Devices, USA). The results were plotted and fitted with GraphPad Prism 8, and the fitted curve was fitted with a 4-parameter nonlinear regression fit to calculate the EC50 value.

Serum competition test (biofilm layer interferometry): collect the second booster immunogen and AddaVax adjuvant equal volume mixed with booster immunized mouse serum, and equal volume (5 μL) of each mouse in the same immunogen group. ) serums were mixed together to characterize the group as a whole. For competition assay experiments, RBD-biotin at concentrations above 5 μg/mL were captured on the biosensor. Then, to saturate the RBD, 2-fold serial dilutions of mouse serum from each group and control group mixed with PBST were loaded onto the biosensor for 300 s. After loading, 400 mM ACE2 or CB6 antibody was combined with the biosensor for 300 s, and the competitive binding signal under the saturation of mouse serum at each dilution level was detected. The sensor was regenerated with 10 mM glycine pH 1.5. Collect real-time signal data to show competitive behavior through different curves of ACE2/CB6 binding signals. Binding signal data was retrieved from the curve, Ro represents the height of the saturated non-competitive binding curve, and Rc represents the saturated competitive binding curve for each dilution level. The relative competition level for each serum dilution level can be calculated as (Ro-Rc)/Rc.

3. Experimental results

The results of this study, shown in Figure 4B, immunization with equal volumes of a dose of a mixture of AddaVax or SAS adjuvant and RBD monomer did not induce RBD-specific total IgG antibodies. Subcutaneous immunization with three RBD-conjugated nanoparticles, RBD-Ferritin, RBD-mi3 or RBD-I53-50, and an equal volume of AddaVax adjuvant induced 71.8 to 168.4-fold greater RBD specificity relative to RBD monomeric protein Total IgG antibodies ( _ED50 : ^103.8±0.4 , ^103.9±0.2 , ^104.2±0.2 , respectively). Similar to Addvax adjuvant, the addition of SAS adjuvant and RBD-Ferritin, RBD-mi3 or RBD-I53-50 granule protein induced mice to produce almost the same antigen-specific binding antibodies (ED ₅₀ : 10 ^4.1±0.3 , 10 respectively). ^{4.0 ± 0.2} , 10 ^{4.3 ± 0.2} ). With the first and second booster immunizations, the total IgG-binding antibodies induced by the addition of AddaVax or SAS adjuvant to RBD-conjugated nanoparticles gradually increased, with a significant upward trend relative to the immunization of RBD monomers (Fig. 4B). To explore the details of the immune response during immunization, we further evaluated the IgG subtype of the elicited antibodies, and the results showed that, regardless of the IgG subtype, the antibody titers showed a similar trend among different immunized groups. In addition, the IgG1:IgG2a binding antibody titer ratio between each group was greater than 1 during the whole immunization process, indicating that the antibody induced mainly Th2 immune response.

In addition to the strength of antibody production, the neutralizing ability of antibody production is another key factor affecting the quality of immunity. Therefore, to further evaluate the immunogenicity of RBD-conjugated nanoparticles, we performed a serum competition assay by the BLI method. The mouse serum after the second booster immunization was taken and mixed in each group for comprehensive evaluation. After serial 2-fold dilution, the serially diluted serum was applied to the RBD captured by the biosensor for blocking. We observed that sera from the RBD-conjugated nanoparticles group preferentially blocked ACE2 and CB6 antibodies from binding to RBD at different dilution levels compared to RBD monomer (Fig. 4C). After recording the binding signal, the height of the non-competitive binding curve Ro and the competitive binding curve Rc of each dilution level can be used for quantitative analysis. A heat map of relative competition levels in mouse serum further showed that competition levels in the RBD-conjugated nanoparticles group were 4-16-fold stronger than in the monomeric RBD group (Fig. 4D). The competition between RBD-Ferritin and RBD-I53-50/mi3 nanoparticles was more intense as the RBD surface copy number increased. The stronger level exhibited by relative competition may indicate that the RBD of the spike protein on the virus is able to invade more persistently and hinder its binding to ACE2 to prevent cell infection, which will be further confirmed by neutralization assays.

Example 6 Determination and comparison of serum neutralization titers induced by immune RBD-conjugated nanoparticles and RBD monomers using SARS-CoV-2 pseudovirus and live virus

To verify the ability of RBD-conjugated nanoparticles to induce neutralizing antibodies in mice, 6-week-old BALB/c mice were subcutaneously immunized with 5 μg of RBD monomer, three RBD-conjugated NPs with an equimolar mass of RBD monomer: RBD- mi3 (9.51μg/beast), RBD-Ferritin (9.34μg/beast) and RBD-I53-50 (11.91μg/beast), PBS was used as a control group, with 5 rats in each group. Booster immunizations were given after a two-week interval, a total of two times. Serum was collected 10 days after each immunization. To assess serum neutralizing antibody titers induced by immuno-RBD-conjugated nanoparticles, we utilized SARS-CoV-2 pseudovirus, live virus-induced cytopathic effect (CPE) and focus reduction neutralization assays (Focus Reduction Neutralization). Test, FRNT).

SARS-CoV-2 pseudovirus: According to previous reports, SARS-CoV-2 pseudovirus was generated in HEK293T cells. Briefly, HEK293T cells were transfected with PsPAX2, pCMV14-SARS-CoV-2 SΔCT-3×Flag and pLenti-GFP at a mass ratio of 1:2:1 by PEI-MAX, and replaced with fresh complete medium 5 h after transfection. After 64 hours, the supernatant containing the SARS-CoV-2 pseudovirus was harvested, precipitated with PEG ₈₀₀₀ solution, concentrated and stored at -80°C. To detect serum neutralizing antibody titers, 1.75×10 ⁴ HEK293T-hACE2 cells were plated in 96-well cell culture plates 12 h before virus infection. The initial concentration of mouse serum was 1:20, the serum was serially diluted 4-fold in complete medium, an equal volume of SARS-CoV-2 pseudovirus was added, a total of 100 μL, and incubated at 37 °C for 2 h. Serum and virus mixture was added to HEK293T-hACE2 cells for 2 h of infection, cell supernatant was discarded, 48 h after infection, lysate was added and luciferase activity was measured by Dual-Glo Luciferase Assay System (Promega). Cells without mouse serum and virus only were added as negative and positive controls, respectively. The results of the study are shown in Figure 5A. In the serum collected after the second booster immunization, the neutralization titer of the serum collected by RBD-conjugated nanoparticles formulated with AddaVax adjuvant was higher than that of the serum of the RBD monomer control group. About 10 to 120 times higher. Similarly, similar results were obtained when the vaccine was formulated with immunogen and SAS adjuvant.

We further examined the neutralizing activity of live SARS-CoV-2 virus against serum using CPE and focal reduction neutralization assays.

The PRNT ₉₀ :Focus reduction neutralization test method was consistent as previously reported. Briefly, serum was diluted from 1:10, serially diluted 5-fold, mixed with an equal volume of 100 Focus Forming Unit (FFU) SARS-CoV-2 CHN/IQTC01/2020 strains, added to 96 wells Incubate at 37°C for 1 h. The mixture was then added to a 96-well plate pre-seeded with Vero-E6 cells. After 1 hour incubation at 37°C, 5% _CO2 , the mixture was removed and replaced with 100 μL of MEM containing 1.2% carboxymethylcellulose, pre-warmed to 37°C, and incubated for an additional 24 hours. Thereafter, cells were fixed with 4% paraformaldehyde and internalized in PBS containing 0.2% Triton X-100, then incubated with anti-rabbit SARS-CoV-2 nucleocapsid protein antibody (Yiqiao Shenzhou) for 1 hour at room temperature, after which Horseradish peroxidase-conjugated 1:4000 diluted goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) was added. Stain using TrueBlue ^™ peroxidase substrate (KPL). Focus was determined and calculated by means of an ELISPOT plate reader (Cellular Technology Ltd. Cleveland, OH). The 90% neutralizing antibody titer (NT ₉₀ ) was defined as the inverse of the serum dilution that inhibited viral infection by 90% FFU, and the fitted curve was fitted with 4-parameter nonlinear regression using GraphPad Prism 8 and calculated. The results are shown in Figure 5B, the neutralizing antibody titers of the sera collected 10 days after the second booster immunization of the RBD-Ferritin, RBD-mi3 or RBD-I53-50 nanovaccine prepared with AddaVax adjuvant were significantly higher. About 10-40 times higher than RBD monomer. Similar to AddaVax, the FRNT ₉₀ titers of the three SAS-adjuvanted RBD-conjugated nanoparticles were also shown to be significantly higher than those of the RBD monomer (Fig. 4B).

Neutralization assay established by CPE: Serum was initially diluted 1:4, serially diluted 4-fold with DMEM supplemented with 2% FBS and 1% penicillin and streptomycin, and an equal volume of 100 tissue culture infectious dose was added. doses, TCID50) SARS-CoV-2- _XN4276 live virus, incubated at 37°C for 2h. After incubation, the mixture was added to Vero-E6 cells pre-plated in a 96-well culture plate, incubated at 37° C. and 5% carbon dioxide for 96 h, and CPE was observed. Pure virus-treated wells, pure diluted serum-treated wells, or cells only were set up in each dish as controls. Virus back-titration was performed on each plate simultaneously. All diluted serum samples were performed in duplicate. Neutralizing antibody titers for all sera were defined as the reciprocal dilution of sera capable of neutralizing 50% of viral infection 4 days post-infection.

Table 3 Table of SARS-CoV-2 live virus neutralization titers determined according to induced cytopathic effect (CPE)

The results are shown in Table 3. After one dose of immunization, the neutralization effect between each group was compared with each other and it was found that RBD-mi3, RBD-Ferritin and RBD-I53-50 nanoparticles could induce neutralizing antibodies, while immunization with RBD monomers No neutralizing antibodies were induced. With the strengthening of the immunization procedure, regardless of the adjuvant used, the neutralization effect of serum samples in the RBD-conjugated nanoparticles group was significantly better than that in the monomeric RBD group. In particular, the RBD nanoparticle-conjugated group could observe the comparison of serum neutralization activity after the first immunization boost, while the monomeric RBD group only had the same intensity of neutralization after the second immunization boost. The neutralization activity of the particle group was almost 10 times higher. Interestingly, when we performed parallel comparisons between groups, RBD-Ferritin nanoparticles exhibited inferior efficacy relative to the other two nanoparticles, which is consistent with the competition analysis (Table 3).

Example 7 Cellular immunity induced by immunizing RBD-conjugated nanoparticles

To verify the ability of RBD-conjugated nanoparticles to induce T-cell immune responses in mice, mice were euthanized 12 days after immunization three times, and the draining lymph nodes of the mice were collected. In order to simultaneously identify germinal center (GC) B cells and T follicular helper cells (Tfh), draining lymph nodes were prepared as cell suspensions, cells were stained and fixed with viability stain 780, and blocked by CD16/32 antibody. Then use the following antibodies for staining and labeling: anti-B220-BV421 (BD Biosciences), anti-IgD-PE (BD Biosciences), anti-GL7-Alexa Fluor 647 (BD Biosciences), anti-CD95-FITC (BD Biosciences), anti-CD4-BV510 (BD Biosciences), anti-CD44-BV786 (BD Biosciences), anti-ICOS-PE-Cyanine7 (BD Biosciences), anti-CXCR5-PE-CF594 (BD Biosciences) and anti-PD-1- APC-R700 (BD Biosciences). Fluorescence signals from labeled samples were collected using a CytoFLEX S flow cytometer (BECKMAN COULTER). Results As shown in Figures 6A and 6B, no significant differences were observed in Tfh and GC cell numbers in draining lymph nodes collected after three subcutaneous immunizations with RBD-conjugated nanoparticles relative to monomeric RBD.

In order to deeply observe the ability of RBD-conjugated nanoparticles to induce T cell immune response in mice, mice were euthanized 40 days after immunization, and the draining lymph nodes and spleen of mice were collected for intracellular factor staining. Draining lymph nodes and spleen were washed with RPMI 160 to prepare a cell suspension, filtered through a 40 μm nylon mesh, washed, and added with red blood cell lysate (1.5 M NH ₄ Cl, 100 mM NaHCO ₃ , 10 mM EDTA in double distilled water, pH 7.4). After lysis of red blood cells, the cells are centrifuged, washed and counted. Approximately 1.0×10 ⁶ lymphocytes were added to a 6-well plate, anti-CD16/32 antibody was added to block Fc receptors, followed by 15 μg/mL of RBD monomeric protein. After 3 h stimulation, GolgiStop and GolgiPlug (BD Biosciences) were added for additional incubation. 15h, preventing the secretion of intracellular factors into the supernatant. After cell washing, anti-CD3e-PerCP-Cy5.5 (BD Biosciences), anti-CD4-BV510 (BD Biosciences) and anti-CD8a FITC (BD Biosciences) labels were added. Cells were fixed by adding 4% paraformaldehyde and internalized in PBS containing 2% BSA, 0.1% saponin, 0.05% _Na3N . Finally, cells were washed twice and the following antibodies were added: anti-IFN-γ-PE-CY7 (BD Biosciences), anti-IL-2-APC (BD Biosciences), anti-TNF-α-PE (BD Biosciences) and control anti-IgG1 labeling. Fluorescence signals from labeled samples were collected using a CytoFLEX S flow cytometer (BECKMAN COULTER).

The results are shown in Figure 6C, 6D and Figure 7, relative to monomeric RBD, the draining lymph node and spleen CD4, CD8aT cells collected after three subcutaneous immunizations with RBD-conjugated nanoparticles co-expressed IL-2, IFN-γ or TNF- There is no significant difference in α, this result is consistent with the reported results of spleen collected from RBD-SC-dimer immunized mice [Dai, L., et al. A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS . Cell 182, 722-733e711 (2020)].

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

An immunogenic complex, characterized in that it comprises:

a) The nanoparticle carrier obtained by self-assembly of the carrier protein fused and expressed with SpyCatcher;

b) RBD antigen of SARS-CoV-2 virus expressed in fusion with SpyTag;

The carrier protein is selected from mi3 and I53-50;

The carrier protein and the antigen are covalently linked through SpyCatcher-SpyTag;

in:

The amino acid sequence of the RBD antigen is as shown in SEQ ID NO: 1;

The amino acid sequence of the mi3 is shown in SEQ ID NO: 3; the I53-50 protein is assembled from the trimeric I53-50A1.1PT1 and the pentameric I53-50B.4PT1, and the I53-50A1.1PT1 Contains the amino acid sequence shown in SEQ ID NO:4; the I53-50B.4PT1 contains the amino acid sequence shown in SEQ ID NO:5.
The immunogenic complex according to claim 1, wherein the SpyCatcher in the component a) is fused to the carrier protein through a linker peptide;

and / or;

In component b), the SpyTag is fused to the RBD antigen through a linker peptide.
The immunogenic complex according to claim 2, wherein the connecting peptide is a flexible connecting peptide.
The immunogenic complex according to claim 3, wherein the amino acid sequence of the connecting peptide in component a) is GGSGGSGGSGGS.
The immunogenic complex according to any one of claims 1 to 4, wherein the SpyCatcher is located at the N-terminus of the carrier protein.
The immunogenic complex according to any one of claims 1 to 4, wherein the SpyTag is located at the C-terminus of the carrier protein.
The immunogenic complex according to any one of claims 1 to 4, wherein the SpyTag contains the amino acid sequence shown in SEQ ID NO: 6; the SpyCatcher contains the amino acid sequence shown in SEQ ID NO: 7 sequence.
A nanoparticle vaccine comprising the immunogenic complex according to any one of claims 1 to 7.
The nanoparticle vaccine according to claim 8, further comprising a pharmaceutically acceptable carrier and/or adjuvant.
The nanoparticle vaccine according to claim 9, wherein the adjuvant is Sigma Adjuvant Systerm and/or AddaVax.
A complete kit, characterized by comprising the nanoparticle vaccine of any one of claims 8 to 10, and a container for inoculating the nanoparticle vaccine.
The method for preparing an immunogenic complex according to any one of claims 1 to 7, characterized in that it comprises:

The fusion protein in the a) component and the b) component is expressed, purified and co-incubated, and the immunogenic complex obtained by self-assembly.
The application of the immunogenic complex according to any one of claims 1 to 7, or the nanoparticle vaccine according to any one of claims 8 to 10, in the preparation of a drug for the treatment of novel coronavirus pneumonia.