WO2022032829A1 - Nanogel de domaine de liaison au récepteur de protéine de spicule, son procédé de préparation et son utilisation - Google Patents

Nanogel de domaine de liaison au récepteur de protéine de spicule, son procédé de préparation et son utilisation Download PDF

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WO2022032829A1
WO2022032829A1 PCT/CN2020/119008 CN2020119008W WO2022032829A1 WO 2022032829 A1 WO2022032829 A1 WO 2022032829A1 CN 2020119008 W CN2020119008 W CN 2020119008W WO 2022032829 A1 WO2022032829 A1 WO 2022032829A1
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nanogel
rbd
binding domain
virus
receptor binding
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PCT/CN2020/119008
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Chinese (zh)
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林坚
陈鹏
陈龙
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北京大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/36—Oxygen or sulfur atoms
    • C07D207/40—2,5-Pyrrolidine-diones
    • C07D207/404—2,5-Pyrrolidine-diones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • C—CHEMISTRY; METALLURGY
    • 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 present invention relates to the fields of medicine and bioengineering, in particular to a multimer nanogel of a viral spike protein receptor binding domain, a preparation method and application thereof, and in particular, to a multimer nanogel capable of enhancing the spike protein receptor binding domain in the Lymph node enrichment and nanogels for enhanced uptake by antigen presenting cells.
  • the SARS-CoV-2 virus binds to the receptor of the host cell through the receptor binding domain (RBD) on the S protein.
  • the S protein is the most important surface protein of the coronavirus. It is a large type I transmembrane protein containing two subunits, S1 and S2.
  • the RBD is located on the S1 subunit and is responsible for recognizing the cell's receptor, which determines the host range of the virus and Specificity plays a key role in mediating the binding of virions to host cell receptors, inducing neutralizing antibodies, T cell responses, and protective immunity, making them ideal vaccine antigens.
  • the purpose of the present invention is to provide a multimeric nanogel (nanogel) formed by chemical cross-linking of the receptor binding domain of the viral spike protein and its application.
  • the multimeric nanogel can enhance the enrichment of the spike protein receptor binding domain in lymph nodes and enhance the uptake of antigen-presenting cells; and can release S-RBD monomer protein in response to reducing conditions, resulting in a strong and effective immune response .
  • a first aspect of the present invention provides a nanogel, which is obtained by chemical cross-linking of a viral spike protein receptor binding domain and a cross-linked molecule; the structural formula of the cross-linked molecule is shown in formula I :
  • R 1 and R 2 are each independently selected from -CH 2 - or -O-;
  • n and m are each independently selected from integers from 1 to 5, such as 1, 2, 3, 4, 5.
  • the virus is a virus that uses glycoproteins as ligands to bind to cellular receptors.
  • the virus is coronavirus, Ebola virus or respiratory syncytial virus (RSV).
  • the coronavirus is a betacoronavirus, including SARS-CoV-1 (polar respiratory syndrome caused by severe illness), SARS-CoV-2 (2019 novel coronavirus), HCoV-OC43, HCoV-HKU1, MERS-CoV.
  • SARS-CoV-1 polar respiratory syndrome caused by severe illness
  • SARS-CoV-2 2019 novel coronavirus
  • HCoV-OC43 HCoV-HKU1
  • MERS-CoV MERS-CoV.
  • the spike protein in the present invention refers to the spike-like glycoprotein or glycoprotein spike on the surface of the viral capsule, which acts as a ligand to specifically recognize the cell receptor when the virus binds to the cell receptor.
  • the virus is SARS-CoV-2
  • the nanogel of the present invention is composed of the SARS-CoV-2 virus spike protein receptor binding domain (receptor binding domain of SARS-CoV-2) spike protein, S-RBD) and the cross-linked molecule shown in formula I are obtained by chemical cross-linking; the amino acid sequence of the SARS-CoV-2 virus spike protein receptor binding domain is SEQ ID NO: 1.
  • the molar ratio of the cross-linking molecule to the viral spike protein receptor binding domain is 10-50:1, for example, 10:1, 20:1, 30:1, 40:1, 50:1.
  • the particle size of the nanogel is 16-50 nm, preferably 20-40 nm.
  • the average particle size of the nanogel is 20-40 nm, such as 20 nm, 25 nm, 30 nm, 35 nm, 40 nm.
  • a second aspect of the present invention provides a method for preparing the nanogel, comprising: mixing the viral spike protein receptor binding domain and the cross-linked molecule, after incubation, and purifying to obtain the nanogel .
  • the incubation temperature is 20-35° C., preferably 30° C.; the incubation time is 0.5-2 h, preferably 1 h.
  • the purification includes passage through a PD-10 column to remove excess cross-linked molecules.
  • viral spike protein receptor binding domain is obtained by heterologous expression.
  • the heterologous expression includes the following steps: obtaining a gene encoding the viral spike protein receptor binding domain; constructing a host cell capable of expressing the encoded gene; The host cell is cultured under the culture conditions of the domain; the culture product is collected and the viral spike protein receptor binding domain is isolated and purified.
  • the host cells are Escherichia coli, yeast or mammalian cells; preferably yeast, such as Pichia, Saccharomyces cerevisiae, more preferably Pichia.
  • the coding gene conforms to the codon preference of the host cell.
  • the virus is SARS-CoV-2
  • the nucleotide sequence of the encoding gene is SEQ ID NO: 2.
  • the third aspect of the present invention provides the use of the nanogel in the following aspects:
  • the virus is SARS-CoV-2, and the use of the nanogel in the following aspects is provided:
  • the vaccine is a subunit vaccine.
  • a fourth aspect of the present invention provides a vaccine composition comprising the nanogel of the present invention and an acceptable vaccine adjuvant.
  • the vaccine adjuvant is toll-like receptor 1/2 agonist Pam3CSK4.
  • the nanogel provided by the present invention can improve the uptake rate of antigen-presenting cells and induce a more rapid and effective immune response.
  • the nanogel provided by the present invention can be applied to all infection by glycoprotein-binding cell receptors.
  • Cellular viruses including coronaviruses, especially betacoronaviruses such as SARS-CoV-1, SARS-CoV-2, HCoV-OC43, HCoV-HKU1, MERS-CoV, etc., and others that infect cells by binding to cell receptors with glycoproteins.
  • Viruses such as Ebola virus, respiratory syncytial virus, etc.
  • the nanogel provided by the present invention is of great significance for the research and development of SARS-CoV-2 vaccines.
  • SARS-CoV-2 vaccines such as the SARS-CoV-2 whole virus inactivated vaccine from China, have shown efficacy in mice, rats and monkeys; another clinical trial of a recombinant adenovirus vaccine ( NCT 04313127) published Phase 1 results, observing neutralizing antibodies and specific T cell responses.
  • whole virus vaccines are expensive, more dangerous in the production process, and can cause serious vaccine-related illnesses.
  • Virus antigen protein subunit vaccines should be a safer, more effective and more economical strategy. Recombinant expression of this antigen in organisms such as E. coli, yeast or mammalian cells will facilitate large-scale production, thereby benefiting more people.
  • S-RBD receptor-binding domain of the SARS-CoV-2 spike protein mediates viral entry into host cells through interaction with human angiotensin-converting enzyme 2 (hACE2). This makes S-RBD a potential candidate for subunit vaccines.
  • hACE2 human angiotensin-converting enzyme 2
  • the present invention provides a nanogel that can degrade and release S-RBD monomer protein in response to reducing conditions, and the immunogenicity of S protein can be enhanced by the gel.
  • the nanogel can improve lymph node targeting and antigen-presenting cell uptake, and during in vivo immunization, the nanogel can be rapidly converted into S-RBD monomeric protein, resulting in a stronger immune response.
  • the S-RBD nanogel provided by the present invention alone can induce a rapid and effective immune response, which makes the nanogel promising to be developed as a safer subunit vaccine.
  • the present invention also provides a vaccine composition containing an adjuvant, which can further improve the immune response and has a good application prospect.
  • the present invention provides a method for preparing S-RBD nanogel, which can produce S-RBD monomer protein in a large amount and safely through heterologous expression, and use it for subsequent nanogel preparation.
  • the preparation method has the advantages of simple steps, no pollution, good stability and the like.
  • FIG. 1 is a schematic diagram of the principle of the S-RBD nanogel provided by the present invention causing an immune response.
  • Figure 2 shows the results of SDS-PAGE and western blot analysis of recombinantly expressed S-RBD protein.
  • Figure 3 is a schematic diagram of the S-RBD nanogel structure
  • A Schematic diagram of the reaction of preparing nanogels with S-RBD and cross-linking agent
  • Fig. 4 is the particle size analysis result of S-RBD nanogel
  • Figure 5 shows the results of SDS-PAGE analysis of S-RBD nanogels and their degradation under reducing conditions.
  • FIG. 6 Fluorescence confocal microscopy images of DC2.4 cells treated with S-RBD nanogels for 1 h and 24 h; the scale bar shown in the figure is 50 ⁇ m.
  • Figure 7 is an analysis diagram of the results of uptake of S-RBD-NG by DC2.4 cells and RAW 264.7 cells;
  • A Fluorescence confocal microscopy image of S-RBD-NG uptake by DC2.4 cells; the scale bar shown in the figure is 50 ⁇ m;
  • Figure 8 is an experimental analysis diagram of S-RBD-NG enriched in lymph nodes in mice
  • A Schematic diagram of the experimental process of enriching S-RBD-NG in lymph nodes in mice
  • Fig. 9 is the antibody titer detection result after the second round of immunization in mice.
  • Figure 10 shows the results of antibody titer detection in mice after the third round of immunization
  • Figure 11 shows the results of antibody titer detection in mice immunized with S-RBD-NG and Pam3CSK4;
  • Fig. 12 shows the detection results of the interaction between S-RBD and hACE2 detected by competitive ELISA; the horizontal axis represents the fold of serum dilution.
  • Figure 13 is the experimental result of neutralizing SARS-CoV-2 pseudovirus by immune serum
  • Figure 14 is a fluorescence confocal microscope image of SARS-CoV-S1-NG uptake by RAW 264.7 cells; the scale bar shown in the figure is 50 ⁇ m.
  • the gene encoding the SARS-CoV-2 S protein receptor binding domain was artificially synthesized.
  • the nucleotide sequence of the gene is SEQ ID NO: 2, using XhoI/NotI enzymes. Cut the site to connect it to the pPICZ ⁇ A vector, transform it, select bleomycin-resistant clones, extract the plasmid for PCR identification and sequencing identification, and identify the correct recombinant plasmid is the recombinant expression plasmid pPICZ ⁇ A-S-RBD.
  • the positive clones screened in step 3 were added to 2.5mL YPD medium, 28°C, shaker 250rpm and cultured to OD 600 of 2-6, about 16-18h; all of them were transferred to 100mL YPD medium, 28 °C, shake at 250 rpm and culture to OD 600 of 2-6, about 8-12 h; transfer 10 mL to 200 mL of BMGY medium, cultivate at 28 °C, 250 rpm until OD 600 is 1, replace with BMMY medium and resuspend; add The final concentration of methanol was 0.5% to induce expression. Methanol should be added every 24h and purified after 72h.
  • Example 2 The S-RBD protein monomer purified in Example 2 was cross-linked into a multimer by a cross-linking agent, and the schematic structural diagram of the multimer is shown in Figure 3A. Specific steps are as follows:
  • the S-RBD protein monomer prepared in Example 2 was mixed with the cross-linking agent CL1 shown in formula i or the cross-linking agent CL2 shown in formula ii with molar equivalents of 10, 20, and 50, respectively, and incubated at 30 °C for 1 h with continuous shaking. .
  • the reaction mixture was then passed through a PD-10 column to remove excess crosslinker;
  • nanogels with different spacer groups Two kinds of nanogels with different spacer groups were prepared, and the schematic diagrams of the spacer groups are shown in Figure 3C, and both contain a disulfide bond inside.
  • the nanogels were taken up by antigen presenting cells (APCs)
  • the disulfide bonds were reduced, and the nanogels prepared from CL1 (denoted as S-RBD-CL1) and the nanogels prepared from CL2 (denoted as S-RBD) -CL2) can be decomposed to release the S-RBD protein monomer.
  • the protein monomer obtained by the reduction of S-RBD-CL1 has a thiol group
  • the protein monomer obtained by the reduction of S-RBD-CL2 can restore the natural amino group.
  • Example 2 The monomer obtained in Example 2 and the multimer obtained in Example 3 were respectively added to Tris/HCl buffer at pH 9.0, and after thorough mixing, Cy5.5-NHS was added to it, mixed quickly, and put Go to 25°C 1000rpm mixer for overnight reaction (protect from light throughout the process), use desalting column to remove unreacted Cy5.5-NHS, and finally filter with 0.22 ⁇ M filter membrane, the products after the reaction are recorded as S-RBD- Cy5.5, S-RBD-CL1-Cy5.5, S-RBD-CL2-Cy5.5, and stored at 4°C in the dark.
  • S-RBD-CL1-Cy5.5 with different equivalents of CL1 and S-RBD-CL2-Cy5.5 with different equivalents of CL2 prepared in Example 4 were mixed with equal volumes of 2 ⁇ denaturing and non-denaturing loading buffers, respectively. Homogenize, boil at 100 °C for 10 min, and after natural cooling, centrifuge at 12,000 rpm for 1 min, and take 30 ⁇ L of sample and load it into the gel sample well. The concentration of separating gel was 12%, and electrophoresis was performed at a constant voltage of 140 V for 1 h. After electrophoresis, the bands were observed by Coomassie brilliant blue staining and Cy5.5 fluorescence.
  • the uptake of antigen by antigen-presenting cells is the key to antigen processing and cross-presentation. This example verifies the uptake ability of antigen-presenting cells for S-RBD-CL1 and S-RBD-CL2; The uptake capacity of the combined agent and S-RBD for different ratios of nanogels. Specific steps are as follows:
  • DC2.4 cells were treated with S-RBD-Cy5.5 (0.1 nmol) prepared in Example 4, and S-RBD-CL1-Cy5.5 (0.1 nmol) with different equivalents of CL1 prepared in The equivalent CL2 S-RBD-CL2-Cy5.5 (0.1nmol) treated DC2.4 cells respectively. After the co-incubation, washed three times with PBS, added Hoechst (nucleus staining) to stain DC2.4 cells, and then used co-incubation. The surface fluorescence of DC2.4 was observed by focusing microscope.
  • S-RBD nanogels prepared using both CL1 and CL2 aggregated significantly more in DC2.4 cells compared to S-RBD monomer.
  • the S-RBD nanogel formed from CL2 (named S-RBD-NG, and will be used hereinafter) is preferably used for ligation. down for research and experimentation.
  • DC2.4 cells and RAW 264.7 cells were used as the cells to be treated, respectively, and S-RBD-NG (0.1 nmol) with different cross-linking agent/S-RBD molar ratios (10 ⁇ , 20 ⁇ , 50 ⁇ ) were used for the cells according to the above method.
  • S-RBD-NG 0.1 nmol
  • FIG. 7A and C the results of quantitative analysis of the imaging data are shown in Figure 7B and D.
  • the quantitative analysis shows that compared with S-RBD monomer, S-RBD-NG has a
  • the uptake effect is affected by the CL2 equivalent, and the nanogel has the best effect when 50 molar equivalent of CL2 is used, which can enhance the uptake of antigen-presenting cells by about 4 times.
  • Example 7 S-RBD-NG increases lymph node enrichment
  • mice were injected intramuscularly with 0.66 nmol S-RBD-Cy5.5, 10 molar equivalents of CL2 S-RBD-NG-Cy5.5, 50 molar equivalents of CL2 S-RBD-NG-Cy5.5 and so on Amount of Cy5.5, as shown in Figure 8A, mice were killed by cervical dislocation 24 hours after injection, 75% alcohol was sprayed on the surface, the limbs of the mice were fixed on the dissection table with pins, the skin of the mice was cut with scissors, and the skin was peeled off. , carefully look for the mouse to take the inguinal lymph node, remove the lymph node with forceps, and use the Maestro mouse imaging system to image the lymph node.
  • C57BL/6N mice were immunized by intramuscular injection with the following reagents: PBS, S-RBD (50 ⁇ g/mouse), S-RBD+aluminum adjuvant (S-RBD 50 ⁇ g/mouse, aluminum hydroxide 100 ⁇ g/mouse), S-RBD RBD-NG (50 ⁇ g/mouse), S-RBD-NG+aluminum adjuvant (S-RBD-NG 50 ⁇ g/mouse, aluminum hydroxide 100 ⁇ g/mouse).
  • PBS PBS
  • S-RBD 50 ⁇ g/mouse
  • S-RBD+aluminum adjuvant S-RBD 50 ⁇ g/mouse, aluminum hydroxide 100 ⁇ g/mouse
  • S-RBD RBD-NG 50 ⁇ g/mouse
  • S-RBD-NG 50 ⁇ g/mouse, aluminum hydroxide 100 ⁇ g/mouse
  • mice were further boosted with the same dose on days 14 and 28 of the first immunization, and sera were collected one week after each immunization (ie, days 7, 21, 35 after the first immunization).
  • S-RBD-specific serum IgG was detected by enzyme-linked immunosorbent assay (ELISA), and the titer was calculated.
  • IgG titers in all groups remained below the detection limit (below the lowest dilution factor of 50, data not shown).
  • serum IgG titers increased to -104 in the S - RBD-NG treated group in the presence and absence of aluminum adjuvant ( Figures 9A and 9B).
  • Aluminum hydroxide is one of the most commonly used adjuvants in the art, however, according to the results of Example 8, it has no obvious improvement effect on the immunogenicity of S-RBD-NG.
  • the present invention explores various adjuvants and finds that when Pam3CSK4 is used as the adjuvant of S-RBD-NG, the immune titer of S-RBD-NG can be significantly improved.
  • Example 10 S-RBD-NG can induce specific antibodies
  • mice in Examples 8-9 were immunized, serum was collected: 1 week after each immunization, blood was collected from the orbital vein, and the blood samples were placed in an EP tube for 1 h at room temperature, and then centrifuged at 4000 rpm for 10 min at room temperature. The supernatant was used as a serum sample.
  • HRP-conjugated goat anti-human IgG1-Fc secondary antibody (1:5000 dilution) was added to the plate and incubated for 1 h at room temperature. Then, after washing 4 times with PBST, 100 ⁇ L of TMB was added to each well, and 50 ⁇ L of H2SO4 (2N) was added to each well after incubation at room temperature to stop the reaction. Absorbance at 450 nm was measured immediately. The test results are shown in Figure 12.
  • Example 11 S-RBD-NG can neutralize SARS-CoV-2 pseudovirus
  • the immunized mouse serum obtained in Examples 8-9 was used to neutralize the SARS-CoV-2 pseudovirus to test the utility of S-RBD-NG as a pre-antigen for SARS-CoV-2 subunit vaccine.
  • the SARS-CoV-2 pseudovirus used in this example has a spike protein coat and carries a luciferase gene (called spike-PV-Luc) as a reporter gene. Specific steps are as follows:
  • COS7-hACE2 cells COS7 cell line stably expressing hACE2 cells
  • COS7 cell line stably expressing hACE2 were inoculated into 96-well plates at 1:30 and cultured for 24h.
  • spike-PV-Luc pseudoviruses were incubated with serum at different dilutions (1:20, 1:40) on ice for 1 h.
  • the mixture of the pseudovirus and serum was added to the COS7-hACE2 cells, after culturing for 24 hours, the medium was replaced with fresh medium, and then the culture was continued for 24 hours.
  • the cells were collected and lysed, and the fluorescence intensity was detected with a luciferase reporter to determine the transfection efficiency.
  • the neutralizing activity of serum can be evaluated by detecting the transfection efficiency of spike-PV-Luc pseudovirus.
  • the serum of PBS and S-RBD immunized mice had no obvious inhibitory effect; the serum of S-RBD-NG immunized mice could significantly inhibit the transfection efficiency of pseudoviruses in a concentration-dependent manner; whether adding aluminum Adjuvants were not significantly different in this experiment; whereas sera from mice immunized with S-RBD-NG and Pam3CSK4 almost completely inhibited pseudovirus transfection at both dilutions.
  • Example 1-7 the recombinant S1 subunit of SARS-CoV-1 and CL2 were formulated into a multimeric nanogel (named SARS-CoV-S1-NG) in this experimental example, and raw264 .7 Cell uptake was verified.
  • the test results are shown in Figure 14.
  • the intracellular uptake of SARS-CoV-S1 was significantly enhanced compared with that of the S1 protein monomer. This shows that for other coronaviruses or other viruses that infect cells with glycoprotein binding cell receptors, such as Ebola virus, respiratory syncytial virus, etc., the receptor binding domain of the glycoprotein can be prepared by the present invention.
  • the nanogels described above are used to improve the uptake of antigen-presenting cells, induce a more rapid and effective immune response, and develop subunit vaccines on this basis.

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Abstract

L'invention concerne un nanogel de domaine de liaison au récepteur de protéine de spicule. Le nanogel est obtenu par réticulation chimique d'un domaine de liaison au récepteur de protéine de spicule virale et d'une molécule de réticulation représentée par la formule I. Le nanogel décrit peut améliorer de manière significative le ciblage de ganglion lymphatique ainsi que l'absorption de cellules présentatrices d'antigène. Le nanogel selon l'invention peut rapidement être converti en une protéine monomère S-RBD lors d'un processus immunologique in vivo, ce qui permet de générer une réponse immunitaire forte et efficace. Le nanogel selon l'invention est prometteur pour le développement en tant que vaccin sous-unitaire de haute sécurité.
PCT/CN2020/119008 2020-08-12 2020-09-29 Nanogel de domaine de liaison au récepteur de protéine de spicule, son procédé de préparation et son utilisation WO2022032829A1 (fr)

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