WO2022032829A1 - Spike protein receptor binding domain nanogel, preparation method therefor, and application thereof - Google Patents

Abstract

A spike protein receptor binding domain nanogel. The nanogel is obtained by means of chemically cross-linking a viral spike protein receptor binding domain and a cross-linking molecule represented by formula I. The described nanogel can significantly improve lymph node targeting as well as the uptake of antigen-presenting cells. During an in vivo immunological process, the described nanogel can quickly be converted into an S-RBD monomeric protein, thereby generating a strong and effective immune response and being promising for development as as a high-safety subunit vaccine.

Description

Spike protein receptor binding domain nanogel and preparation method and application thereof technical field

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.

Background technique

Since December 2019, a number of cases of pneumonia of unknown cause have been discovered one after another, which has now been confirmed to be an acute respiratory infectious disease caused by a new type of coronavirus SARS-CoV-2 infection. People infected with the virus will experience varying degrees of symptoms, ranging from just a fever or a mild cough, to pneumonia, to more severe and even death. The new coronavirus SARS-CoV-2 has an incubation period of up to 14 days and is highly contagious.

As a new type of disease, the new coronavirus is still poorly studied. There is currently no effective treatment. Taking preventive measures to stop the spread of the virus is the key to controlling the epidemic. Vaccination is an irreplaceable means of effectively eliminating infectious diseases. Therefore, the rapid development of preventive vaccines that can enhance the level of herd immunity and block the spread of the virus has become the most urgent need at present. The published results of the SARS-CoV-2 virus genome alignment show that the differences between the viruses are very small, and no mutation has been found so far. Therefore, if the SARS-CoV-2 vaccine is successfully developed, it will be able to suppress the outbreak of new epidemics to a large extent.

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.

SUMMARY OF THE INVENTION

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 .

To this end, 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 :

in,

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.

In a preferred embodiment, the structural formula of the cross-linked molecule is shown in formula i or formula ii:

Further, the virus is a virus that uses glycoproteins as ligands to bind to cellular receptors.

Further, the virus is coronavirus, Ebola virus or respiratory syncytial virus (RSV).

In a specific embodiment, 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.

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.

In a preferred embodiment, the virus is SARS-CoV-2, and 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.

Further, 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.

Further, the particle size of the nanogel is 16-50 nm, preferably 20-40 nm. In a specific embodiment, 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 .

Further, the incubation temperature is 20-35° C., preferably 30° C.; the incubation time is 0.5-2 h, preferably 1 h.

Further, the purification includes passage through a PD-10 column to remove excess cross-linked molecules.

Further, the viral spike protein receptor binding domain is obtained by heterologous expression.

Further, 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.

Further, the host cells are Escherichia coli, yeast or mammalian cells; preferably yeast, such as Pichia, Saccharomyces cerevisiae, more preferably Pichia.

Further, the coding gene conforms to the codon preference of the host cell.

In a preferred embodiment, the virus is SARS-CoV-2, and 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:

(1) preparing a vaccine of the virus;

(2) preparing an immune-enhancing drug for the virus.

In a preferred embodiment, the virus is SARS-CoV-2, and the use of the nanogel in the following aspects is provided:

(1) Preparation of SARS-CoV-2 vaccine;

(2) Preparation of immune-enhancing drugs for SARS-CoV-2.

Further, 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.

Further, 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. According to its action principle, 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. In particular, the nanogel provided by the present invention is of great significance for the research and development of SARS-CoV-2 vaccines.

Currently developed 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. However, 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.

The receptor-binding domain (S-RBD) 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. However, the poor pharmacokinetics and low immunogenicity of S-RBD have greatly hindered its development for subunit vaccines. An important reason for the low immunogenicity of S-RBD is its poor targeting to lymph nodes, which are critical for antigen uptake and processing by DCs and macrophages.

Compared with the prior art, the technical solution of the present invention has the following advantages:

(1) 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.

(2) In the absence of adjuvant, 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.

(3) The present invention also provides a vaccine composition containing an adjuvant, which can further improve the immune response and has a good application prospect.

(4) 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.

Description of drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are for the purpose of illustrating preferred embodiments only and are not to be considered limiting of the invention. Also, the same components are denoted by the same reference numerals throughout the drawings. In the attached image:

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;

B: Schematic diagram of the molecular structure of the two cross-linking agents;

C: Schematic illustration of the fracture of nanogels prepared from CL1 and CL2 in response to reducing environment.

Fig. 4 is the particle size analysis result of S-RBD nanogel;

A: Dynamic scattered light analysis (DLS) results of S-RBD nanogels;

B: Transmission electron microscope (TEM) image of the S-RBD nanogel; scale bar shown in the figure is 10 nm.

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 μm.

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 μm;

B: Quantitative analysis results of S-RBD-NG uptake by DC2.4 cells;

C: Fluorescence confocal microscopy image of S-RBD-NG uptake by RAW 264.7 cells; the scale bar shown in the figure is 50 μm;

D: Quantitative analysis of S-RBD-NG uptake by RAW 264.7 cells.

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;

B: Fluorescence imaging image of mouse somatic lymph node;

C: Fluorescence quantitative analysis results of mouse somatic lymph nodes;

D: Uptake analysis of S-RBD-NG and S-RBD by DC cells and macrophages in lymph nodes.

Fig. 9 is the antibody titer detection result after the second round of immunization in mice;

A: The detection results of S-RBD-specific serum IgG in mouse serum by ELISA;

B: Analysis of S-RBD-specific serum IgG titers calculated from A.

Figure 10 shows the results of antibody titer detection in mice after the third round of immunization;

A: The detection results of S-RBD-specific serum IgG in mouse serum by ELISA;

B: Analysis of S-RBD-specific serum IgG titers calculated from A.

Figure 11 shows the results of antibody titer detection in mice immunized with S-RBD-NG and Pam3CSK4;

A: The detection results of S-RBD-specific serum IgG in the serum of mice after the second and third rounds of immunization by ELISA;

B: Analysis of S-RBD-specific serum IgG titers calculated from A.

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;

A: Inhibitory effect of different titers of immune sera on spike-PV-Luc transfection;

B: Inhibitory effect of immune serum on spike-PVGFP transfection.

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 μm.

detailed description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure will be more thoroughly understood, and will fully convey the scope of the present disclosure to those skilled in the art.

Example 1 Construction of Pichia pastoris expression vector of S-RBD

According to the codon preference of Pichia pastoris, the gene encoding the SARS-CoV-2 S protein receptor binding domain (S-RBD) 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 pPICZαA 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 pPICZαA-S-RBD.

Example 2 Expression and purification of S-RBD protein

1. Plasmid linearization

Take 20 μg of pPICZαA-S-RBD plasmid, digest it with SacⅠ, 37 ℃ water bath, and use 1% agarose gel electrophoresis to identify whether it is linearized; after confirming the linearization, add 1/10 volume of acetic acid Sodium, 2 times the volume of absolute ethanol, invert and mix; centrifuge at 14,000 rpm for 15 min at 4°C, carefully remove the supernatant; add 500 μL of 75% ethanol to wash the plasmid pellet, centrifuge at 4°C, 14,000 rpm for 15 min, carefully remove the supernatant, and air dry, Add 30 μL of sterile water to resuspend the linearized plasmid.

2. Plasmid electroporation

Add 10 μL of linearized plasmid to 90 μL of X-33 yeast competent, and add it to the electrode cup after mixing; put the electrode cup into the electroporator, 2000V, 5ms, and perform electric shock; immediately after clicking, add 800 μL of 1M sorbitol to remove it All were aspirated and transferred to a 15mL centrifuge tube, incubated at 25°C with a shaker at 200rpm for 2h; then added 3mL of YPD-free medium, incubated at 25°C with a shaker at 200rpm for 2h; centrifuged at 1100rpm at room temperature, removed the supernatant, and resuspended the bacteria with 300μL YPD. solution, add 100 μL to YPD plate containing bleomycin; incubate at 28°C upside down for 2-3 days.

3. Small batch screening of positive clones

Pick a single clone and add it to 2.5mL YPD medium, 28°C, shaker at 250rpm and culture to OD ₆₀₀ of 2-6, about 16-18h; transfer 150μL to 3mL BMGY medium with pH 6.5, shake at 28°C Incubate at 250 rpm until OD ₆₀₀ is 1, about 8-12 h; change to BMMY medium, add final concentration of 0.5% methanol to induce expression, methanol needs to be added every 24 h, and collect supernatant after 72 h to identify positive clones by SDS-PAGE.

4. Mass induction of positive clones

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 ℃, 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 ℃, 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.

5. Protein purification

Centrifuge the bacterial solution obtained in step 4 at 12,000 rpm for 20 min, and take the supernatant; use 1M Tris to adjust the pH of the supernatant to 8.0; centrifuge at 12,000 rpm, take the supernatant, and filter it with filter paper; the filtered supernatant is purified by AKTA purification system with Ni column. Purification; SDS-PAGE identification was carried out after purification, as shown in Figure 2, after purification, a SARS-CoV-2 S protein receptor binding domain protein with a molecular weight of about 30kDa can be obtained, denoted as S-RBD.

Example 3 Chemically cross-linked S-RBD protein monomers are multimers

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;

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. When 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. As shown in Fig. 3C, the protein monomer obtained by the reduction of S-RBD-CL1 has a thiol group, and the protein monomer obtained by the reduction of S-RBD-CL2 can restore the natural amino group.

Dynamic scattering light analysis (DLS) and transmission electron microscopy (TEM) imaging of the nanogels prepared with 50 molar equivalents of CL2 were performed, and the results are shown in Figures 4A and 4B. According to this measurement, the cross-linked nanogels formed an average diameter of about 25 nm, while the diameter of native S-RBD was about 2 nm as measured by DLS.

Example 4 Monomer and multimer labeled Cy5.5 fluorescence

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 0.22μM 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.

Example 5 Identification by SDS-PAGE

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 μL 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 results are shown in Fig. 5, wherein the denatured band is shown as a monomer with a molecular mass of about 30kDa, and the non-denatured band is of a multimer nature with a size of about 22kDa. This result indicates that the nanogels formed from CL2 show higher efficiency.

Example 6 Uptake by antigen presenting cells

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.

As shown in Figure 6, S-RBD nanogels prepared using both CL1 and CL2 aggregated significantly more in DC2.4 cells compared to S-RBD monomer. Considering that the nanogel prepared from CL2 can regenerate the native S-RBD protein, 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. After processing, the fluorescence observation results are shown in Figure 7A and C, and 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

C57BL/6N 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.

It can be seen from Figure 8B that S-RBD-NG-Cy5.5 has higher aggregation and longer residence time in mouse lymph nodes than S-RBD-Cy5.5, indicating that the multimer prepared by the present invention can significantly increase lymph nodes enrichment. Further quantitative analysis (Fig. 8C) showed that the accumulation of S-RBD-NG with 50 molar equivalents of CL2 increased approximately 3.9-fold compared to S-RBD.

After the lymph nodes were digested into single cells, the uptake of S-RBD-NG and S-RBDCY5 by DC cells and macrophages was analyzed by flow cytometry. The results are shown in Fig. 8D, and it can be seen that the uptake of S-RBD-NG by DC cells and macrophages is significantly higher than that of S-RBD.

Example 8 Immunogenicity test

This example tested the immunogenicity of S-RBD-NG in vivo. Specific steps are as follows:

C57BL/6N mice were immunized by intramuscular injection with the following reagents: PBS, S-RBD (50μg/mouse), S-RBD+aluminum adjuvant (S-RBD 50μg/mouse, aluminum hydroxide 100μg/mouse), S-RBD RBD-NG (50 μg/mouse), S-RBD-NG+aluminum adjuvant (S-RBD-NG 50 μg/mouse, aluminum hydroxide 100 μg/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. One week after the first immunization, IgG titers in all groups remained below the detection limit (below the lowest dilution factor of 50, data not shown). After the second round of immunization, 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). After the third round of immunization, the titers of the S-RBD-NG treated group reached ^-105 , while the titers of the S-RBD monomer treated group were less than ¹⁰⁴ (Figures 10A and 10B). Quantitative analysis showed that the titers induced by S-RBD-NG were 27.6 times higher (without aluminum adjuvant) and 8.3 times higher than that of S-RBD monomer (with aluminum adjuvant); S-RBD-NG had more potency than S-RBD. High immunogenicity, can elicit a more effective and rapid immune response.

Example 9 Enhanced immunogenicity with Pam3CSK4 as adjuvant

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.

In this example, according to the method of Example 8, it was tested that the toll-like receptor 1/2 agonist Pam3CSK4 (5nmol per injection) combined with S-RBD-NG can induce a more effective immune response, see Fig. 11A and 11B, S-RBD-NG ^- specific IgG titers reached -106 after the third round of immunization. This suggests that the immune titer of S-RBD-NG can be further improved by optimizing the use of adjuvants.

Example 10 S-RBD-NG can induce specific antibodies

Since blocking the interaction between the spike protein and ACE2 is essential to prevent SARS-CoV-2 from entering host cells, this example tested whether sera from mice immunized with S-RBD-NG could inhibit this interaction effect. Specific steps are as follows:

After the 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.

Competitive ELISA: S-RBD was diluted with PBS to 1 μg/mL, spread on EIA plates at 4°C, and left overnight. Wash once with PBST (0.5% Tween-20). It was then blocked with 2% BSA in PBS for 2 h. Serum was diluted with PBST-BSA (0.5% Tween, 0.5% BSA), and the plate was pre-blocked with 50 μL serum of different dilutions for 30 min (the control group without serum blocking was set at the same time), and 50 μL ACE-hFc (1 μg/mL) was added. mL) and cultured for another 1 h. It was then washed 3 times with PBST. 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 μL of TMB was added to each well, and 50 μL 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.

It can be seen from Figure 12 that the serum obtained by immunization with S-RBD-NG can effectively block the interaction between S-RBD and hACE2. This shows that the S-RBD-NG prepared by the present invention induces specific antibodies in vivo, which can target and block the interaction between S-RBD and hACE2.

Example 11 S-RBD-NG can neutralize SARS-CoV-2 pseudovirus

In this example, 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) were inoculated into 96-well plates at 1:30 and cultured for 24h. Then, spike-PV-Luc pseudoviruses were incubated with serum at different dilutions (1:20, 1:40) on ice for 1 h. Then, 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. After the incubation, 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. According to the results in Figure 13A, 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.

From the results in Figure 13A, it was found that the transfection efficiency of the pseudovirus was enhanced when using a higher concentration (1:20 dilution) of serum compared to using a lower concentration (1:40 dilution) serum. This may be due to the fact that SARS-CoV-2 can promote viral entry after infection with antibodies in blood (ie, antibody-dependent enhancement). To further confirm this result, another spike pseudovirus (named spike-PVGFP) carrying the gfp gene was prepared in this example. According to the aforementioned experimental method, the serum with a dilution of 1:20 was tested for the efficiency of Spike PV-GFP transfection into COS7-hACE2 cells. The imaging results of confocal microscopy are shown in Figure 13B. The results in Figure 13B show that the sera of mice immunized with S-RBD-NG can effectively neutralize the SARS-CoV-2 pseudovirus.

Example 12

Using a method similar to 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. After the preparation of SARS-CoV-S1 as a nanogel, 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.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. Substitutions should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (10)

A nanogel is characterized in that, it is obtained by chemical cross-linking by the viral spike protein receptor binding domain and cross-linking molecule; the structural formula of the cross-linking molecule is shown in formula I:

in, R ₁ and R ₂ are each independently selected from -CH ₂ - or -O-; n and m are each independently selected from an integer of 1-5. The nanogel of claim 1, wherein the structural formula of the cross-linked molecule is as shown in formula i or formula ii:

Preferably, the virus is a virus that uses glycoproteins as ligands to bind to cellular receptors; more preferably, the virus is coronavirus, Ebola virus or respiratory syncytial virus; more preferably, the coronavirus is Betacoronavirus; preferably, the betacoronavirus includes SARS-CoV-1, SARS-CoV-2, HCoV-OC43, HCoV-HKU1, MERS-CoV. The nanogel according to claim 1 or 2, wherein the molar ratio of the cross-linked molecule to the viral spike protein receptor binding domain is 10-50:1. The nanogel according to claim 1 or 2, wherein the particle size of the nanogel is 16-50 nm. The preparation method of the nanogel according to any one of claims 1 to 4, characterized in that it comprises the following steps: mixing the viral spike protein receptor binding domain with the cross-linked molecule, incubating, and purifying to obtain the nanogel. The preparation method of claim 5, wherein the incubation temperature is 20-35°C; the incubation time is 0.5-2h; Preferably, the purification comprises passage through a PD-10 column to remove excess cross-linked molecules. The preparation method of claim 5, wherein the viral spike protein receptor binding domain is obtained by heterologous expression; Preferably, the heterologous expression comprises the following steps: obtaining a gene encoding the viral spike protein receptor binding domain; constructing a host cell capable of expressing the encoded gene; Culturing the host cell under the culture conditions of the domain; collecting the culture product and isolating and purifying the viral spike protein receptor binding domain; Preferably, the host cells are Escherichia coli, yeast or mammalian cells; Preferably, the encoding gene conforms to the codon preference of the host cell; Preferably, the virus is SARS-CoV-2, and the nucleotide sequence of the encoding gene is SEQ ID NO: 2. Use of the nanogel according to any one of claims 1 to 4 in the following aspects: (1) preparing a vaccine of the virus; (2) preparing the immune-enhancing drug of the virus; Preferably, the vaccine is a subunit vaccine. A vaccine composition, characterized in that it comprises the nanogel according to any one of claims 1 to 4 and an acceptable vaccine adjuvant. The vaccine composition of claim 9, wherein the vaccine adjuvant is Pam3CSK4.

Images