WO2022088953A1 - Vaccin à nanoparticules conjuguées au rbd du sars-cov-2 - Google Patents
Vaccin à nanoparticules conjuguées au rbd du sars-cov-2 Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the invention relates to the field of immunomedicine, in particular to a SARS-CoV-2 RBD conjugated nanoparticle vaccine.
- 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.
- the S protein is the major antigenic component responsible for inducing host immune responses, neutralizing antibodies and protective immunity against coronavirus infection.
- 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.
- the present invention relates to an immunogenic complex comprising:
- nanoparticle carrier obtained by self-assembly of the carrier protein fused and expressed with SpyCatcher;
- the carrier protein is selected from mi3 and I53-50;
- the carrier protein and the antigen are covalently linked through SpyCatcher-SpyTag;
- 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.
- 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.
- 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.
- Fig. 1 is the structure and structural characteristics of RBD-coupled nanoparticles in an embodiment of the present invention
- FIG. 2 is the verification of the assembly properties and physical characteristics of the assembled particles in one embodiment of the present invention.
- Fig. 3 is the antigenic identification of RBD monomer and RBD-conjugated nanoparticle in one embodiment of the present invention
- Fig. 4 is the immunogenicity identification of RBD monomer and RBD-conjugated particle in one embodiment of the present invention
- Fig. 5 is immune serum neutralization experiment in one embodiment of the present invention.
- 6 is the immune cell characteristics of the draining lymph nodes of the mice after each immunization in an embodiment of the present invention
- 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;
- Cytokine-releasing CD4+ cells were identified by co-expressing IFN- ⁇ , IL2 or TNF- ⁇ as markers, and no significant differences were found between the groups;
- Figure 7 shows the immune cell characteristics in the spleen of mice after each immunization
- Cytokine-releasing CD4+ (A) cells were identified by co-expressing IFN- ⁇ , IL2 or TNF- ⁇ as markers, and no significant difference was found between the groups;
- B Cytokine-releasing CD8+ T (B) cells were identified by co-expressing IFN- ⁇ , IL2 or TNF- ⁇ as markers, and no significant differences were found between the groups.
- the present invention relates to an immunogenic complex comprising:
- nanoparticle carrier obtained by self-assembly of the carrier protein fused and expressed with SpyCatcher;
- the carrier protein is selected from Ferritin, mi3 and I53-50;
- the carrier protein and the antigen are covalently linked through SpyCatcher-SpyTag;
- 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.
- 20 trimers I53-50A1.1PT1 and 12 pentamers I53-50B.4PT1 are assembled in vitro with a molar mass ratio of 1:1-3.
- the SpyCatcher in component a) is fused to the carrier protein via a linker peptide.
- the SpyTag in component b) is fused to the RBD antigen via a linker peptide.
- 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.
- 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.).
- the linker peptide is a flexible linker peptide.
- the amino acid sequence of the linking peptide is selected from one or more of Gly, Ser, Pro, Ala, and Glu.
- 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.
- (GGS)n means that there are n GGS repeats, for example (GGS) 4 means GGSGGSGGSGGS, and the same is true for others.
- the amino acid sequence of the linking peptide in component a) is from (GGS) 4 .
- the amino acid sequence of the linking peptide in component b) is GSGGSGGSG.
- the SpyCatcher is N-terminal to the carrier protein.
- the SpyTag is C-terminal to the carrier protein.
- the SpyTag contains the amino acid sequence set forth in SEQ ID NO:6.
- the SpyCatcher contains the amino acid sequence set forth in SEQ ID NO:7.
- component b) is ⁇ N1-SpyCatcher-Ferritin, which contains the amino acid sequence set forth in SEQ ID NO: 9; or
- the component is ⁇ N1-SpyCatcher-mi3, which contains the amino acid sequence shown in SEQ ID NO: 10; or
- the component is ⁇ N1-SpyCatcher-I53-50, which contains the I53-50 protein assembled from the trimeric ⁇ N1-SpyCatcher-I53-50A1.1PT1 and the pentameric I53-50B.4PT1, wherein ⁇ N1 - 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.
- the present invention also relates to a nanoparticle vaccine comprising the immunogenic complex as described above.
- 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
- 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.
- 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.
- Sigma Adjuvant Systerm 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.
- the vaccine is a water-in-oil emulsion having an aqueous phase and an oily phase.
- 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. .
- SC subcutaneous
- IM intramuscular
- IV intravenous
- IP intraperitoneal
- ID intradermal
- 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.
- 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 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.
- 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.
- one or more administrations are administered.
- 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.
- 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.
- 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.
- 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 Wang-Hu-1, GenBank: MN908947
- RBD gene (residues: 319-541) was optimized and synthesized by mammalian codon bias.
- 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.
- the HRV 3C site and the His tag of 6 histidines were fused to the C-terminus of the SpyTag gene.
- 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).
- the ⁇ N1-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 ⁇ N1-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.
- 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).
- 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.
- S protein receptor binding region gene SEQ ID NO: 17
- 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 Expression vector
- VRC8400-RBD-SpyTag-8*His VRC8400-RBD-8*His
- VRC8400-hACE2-8*his 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.
- 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.
- 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.
- 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.
- RBD protein and three nanoparticles ⁇ N1-SpyCatcher-Ferritin, ⁇ N1-SpyCatcher-mi3, ⁇ N1-SpyCatcher-I53-50, and three nanoparticles were expressed and purified, respectively.
- Reagents and consumables 300 mesh copper mesh, disposable solvent-resistant micro-test tubes, etc. are commonly used commercial reagents and consumables.
- 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 ⁇ N1-SpyCatche-NPs protein.
- Table 1 shows the size and distribution of the hydrated particle size of the RBD-conjugated nanoparticle protein and the ⁇ N1-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
- RBD-I53-50 was 50.67 ⁇ 0.11 nm.
- the RBD-conjugated nanoparticle protein was larger, indicating that the RBD protein was displayed on the outside of the ⁇ N1-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).
- 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.
- 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.
- CB6 antibody 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.
- 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 ⁇ g/mL, coated with ELISA plates, 100 ng/well, with a total volume of 100 ⁇ L, 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.
- 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 ⁇ g/mL, coated on ELISA plates, 100 ng/well, with a total volume of 100 ⁇ L, 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.
- Step 1 Calculate the amount of biotinylation reagent
- 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 ⁇ L) for 30 minutes at room temperature.
- 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 ⁇ L of PBS, respectively.
- ACE/CB6-biotinylated protein diluted in buffer was captured on SA sensors at 2 ⁇ g/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.
- 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.
- 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.
- 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.
- mice female BALB/c mice of 6-8 weeks old.
- Adjuvants commercial Sigma Adjuvant Systerm (SAS, Sigma) and AddaVax adjuvant (InvivoGen).
- mice 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.
- the immunization dose was 5 ⁇ g/unit of RBD monomer, and three RBD-conjugated NPs with the same molar mass as RBD monomer: RBD-mi3 (9.51 ⁇ g/unit), RBD-Ferritin (9.34 ⁇ g/unit) and RBD-I53-50 (11.91 ⁇ g/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 ⁇ g/mL, coated with 96-well high-binding ELISA plate, 100 ⁇ L/well, and placed at 4°C overnight; configure the blocking solution as above, add the blocking solution to the ELISA plate , 100 ⁇ L/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
- 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 ⁇ L) of each mouse in the same immunogen group. ) serums were mixed together to characterize the group as a whole.
- RBD-biotin at concentrations above 5 ⁇ g/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.
- 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
- mice 6-week-old BALB/c mice were subcutaneously immunized with 5 ⁇ g of RBD monomer, three RBD-conjugated NPs with an equimolar mass of RBD monomer: RBD- mi3 (9.51 ⁇ g/beast), RBD-Ferritin (9.34 ⁇ g/beast) and RBD-I53-50 (11.91 ⁇ g/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.
- SARS-CoV-2 pseudovirus was generated in HEK293T cells. Briefly, HEK293T cells were transfected with PsPAX2, pCMV14-SARS-CoV-2 S ⁇ CT-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.
- 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 ⁇ L, 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).
- 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 ⁇ L of MEM containing 1.2% carboxymethylcellulose, pre-warmed to 37°C, and incubated for an additional 24 hours.
- FFU Focus Forming Unit
- 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.
- 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.
- mice were euthanized 12 days after immunization three times, and the draining lymph nodes of the mice were collected.
- draining lymph nodes were prepared as cell suspensions, cells were stained and fixed with viability stain 780, and blocked by CD16/32 antibody.
- 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 ⁇ m 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.
- red blood cell lysate 1.5 M NH 4 Cl, 100 mM NaHCO 3 , 10 mM EDTA in double distilled water, pH 7.4
- lymphocytes were added to a 6-well plate, anti-CD16/32 antibody was added to block Fc receptors, followed by 15 ⁇ g/mL of RBD monomeric protein.
- GolgiStop and GolgiPlug (BD Biosciences) were added for additional incubation. 15h, preventing the secretion of intracellular factors into the supernatant.
- anti-CD3e-PerCP-Cy5.5 (BD Biosciences)
- anti-CD4-BV510 BD Biosciences
- anti-CD8a FITC FITC
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Abstract
La présente invention concerne un vaccin à nanoparticules conjuguées au RBD du SARS-CoV-2, comprenant : a) un support de nanoparticules obtenu par auto-assemblage d'une protéine porteuse exprimée en fusion avec SpyCatcher ; b) un antigène du RBD du virus du SARS-CoV-2 exprimé en fusion avec SpyTag, la protéine porteuse étant choisie parmi mi3 et I53-50, et la protéine porteuse et l'antigène étant liés de manière covalente par SpyCatcher-SpyTag.
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