CN112125832B

CN112125832B - Spike protein receptor binding domain nanogel and preparation method and application thereof

Info

Publication number: CN112125832B
Application number: CN202010808232.5A
Authority: CN
Inventors: 林坚; 陈鹏; 陈龙
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2022-09-20
Anticipated expiration: 2040-08-12
Also published as: WO2022032829A1; CN112125832A

Abstract

The invention relates to a spike protein receptor binding domain nanogel which is obtained by chemically crosslinking a virus spike protein receptor binding domain and a crosslinking molecule shown in a formula I. The nanogel can obviously improve lymph node targeting and the uptake of antigen presenting cells, and can be quickly converted into S-RBD monomer protein in the in-vivo immune process, so that strong and effective immune response is generated, and the nanogel has the prospect of being developed into a subunit vaccine with high safety.

Description

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

Technical Field

The invention relates to the field of medicine and bioengineering, in particular to a virus spike protein receptor binding domain polymer nanogel and a preparation method and application thereof, and particularly relates to a nanogel capable of enhancing the enrichment of a spike protein receptor binding domain in lymph nodes and enhancing the uptake of antigen presenting cells.

Background

As a novel disease, the research on the novel coronavirus is shallow. No effective therapeutic medicine exists before the day. The prevention is well done, and the blocking of the spread of the virus is the key to controlling the epidemic situation. Vaccination is an irreplaceable means of effectively eliminating infectious diseases. Therefore, the rapid development of preventive vaccines capable of improving the immunity level of the population and blocking the spread of viruses has become the most urgent need at present. Published results of alignment of SARS-CoV-2 virus genomes show that the difference between viruses is very small and no variation has been found at present. Therefore, if the SARS-CoV-2 vaccine is successfully developed, the outbreak of new epidemic situation can be inhibited to a great extent.

The SARS-CoV-2 virus binds to a receptor of a host cell via a Receptor Binding Domain (RBD) on the S protein. The S protein is the most important surface protein of coronavirus, is a large type I transmembrane protein containing two subunits, S1 and S2, is positioned on the S1 subunit, is responsible for recognizing a receptor of a cell, determines the host range and specificity of the virus, plays a key role in mediating the combination of virus particles and the receptor of the host cell, inducing neutralizing antibodies, T cell reaction and protective immunity, and is an ideal vaccine antigen.

Disclosure of Invention

The invention aims to provide a polymer nanogel (nanogel) formed by chemically crosslinking a receptor binding domain of a virus spike protein and application thereof. The polymer nanogel can enhance the enrichment of the receptor binding domain of the spike protein in lymph nodes and enhance the uptake of antigen presenting cells; and can respond to the reduction condition to release S-RBD monomer protein, so as to cause strong and effective immune response.

To this end, a first aspect of the invention provides a nanogel obtained by chemically cross-linking a viral spike protein receptor binding domain with a cross-linking molecule; the structural formula of the cross-linking molecule is shown as formula I:

wherein the content of the first and second substances,

R ₁ and R ₂ Each independently selected from-CH ₂ -or-O-;

n and m are each independently selected from integers of 1 to 5, such as 1, 2, 3, 4, 5.

In a preferred embodiment, the crosslinking molecule has a structural formula as shown in formula i or formula ii:

further, the virus is a virus that binds to a cell receptor using a glycoprotein as a ligand.

Further, the virus is a coronavirus, an ebola virus, or a Respiratory Syncytial Virus (RSV).

In a specific embodiment, the coronavirus is a beta coronavirus, including SARS-CoV-1 (Severe acute respiratory syndrome), SARS-CoV-2(2019 New coronavirus), HCoV-OC43, HCoV-HKU1, MERS-CoV.

The spike protein of the present invention refers to a spike glycoprotein or glycoprotein spike on the surface of a viral envelope, which serves as a ligand to specifically recognize a cellular receptor when the virus binds to the cellular receptor.

In a preferred embodiment, the virus is SARS-CoV-2, the nanogel of the invention is obtained by chemically crosslinking a SARS-CoV-2 virus spike protein receptor binding domain (S-RBD) and a crosslinking molecule shown in formula I; the amino acid sequence of the SARS-CoV-2 virus spike protein receptor binding structure 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, such as 10:1, 20:1, 30:1, 40:1, 50: 1.

Further, the particle size of the nanogel is 16-50 nm, and preferably 20-40 nm. In a specific embodiment, the nanogel has an average particle size of 20 to 40nm, for example, 20nm, 25nm, 30nm, 35nm, or 40 nm.

In a second aspect of the present invention, there is provided a method for preparing the nanogel, comprising: and mixing the virus spike protein receptor binding domain with the cross-linking molecule, incubating, and purifying to obtain the nanogel.

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

Further, the purification comprises passing through a PD-10 column to remove excess cross-linking molecules.

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

Further, the heterologous expression comprises the steps of: obtaining coding genes of a receptor binding domain of the virus spike protein; constructing a host cell capable of expressing the encoding gene; culturing said host cell under culture conditions suitable for expression of said viral spike protein receptor binding domain; collecting the culture product and separating and purifying the virus spike protein receptor binding structural domain.

Further, the host cell is escherichia coli, yeast or mammalian cell; preferably a yeast, such as pichia, saccharomyces, more preferably pichia.

Further, the encoding 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 coding gene is SEQ ID NO: 2.

in a third aspect of the invention, there is provided the use of the nanogel in:

(1) preparing a vaccine of said virus;

(2) preparing the immune enhancing medicine of the virus.

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

(1) preparing SARS-CoV-2 vaccine;

(2) preparing the immune enhancing medicine of SARS-CoV-2.

Further, the vaccine is a subunit vaccine.

In a fourth aspect of the invention, there is provided a vaccine composition comprising a nanogel according to the invention and an acceptable vaccine adjuvant.

Further, the vaccine adjuvant is a toll-like receptor 1/2 agonist Pam3CSK 4.

The nanogel provided by the invention can improve the uptake rate of antigen presenting cells and induce a quicker and more effective immune response, and according to the action principle, the nanogel provided by the invention can be suitable for all viruses infecting cells by glycoprotein-bound cell receptors, including coronaviruses, particularly beta coronaviruses such as SARS-CoV-1, SARS-CoV-2, HCoV-OC43, HCoV-HKU1, MERS-CoV and the like, and other viruses infecting cells by glycoprotein-bound cell receptors such as Ebola virus, respiratory syncytial virus and the like. Especially, the nanogel provided by the invention has great significance for the research and development of SARS-CoV-2 vaccines.

SARS-CoV-2 vaccines that have been developed so far, such as SARS-CoV-2 whole virus inactivated vaccine from China, have been shown to be effective in mice, rats and monkeys; another recombinant adenovirus vaccine clinical trial (NCT 04313127) published phase 1 results and observed neutralizing antibodies and specific T cell responses. However, whole virus vaccines are expensive, more dangerous in the manufacturing process, and may cause serious vaccine-related diseases. A viral antigenic protein subunit vaccine should be a safer, more effective, and more economical strategy. Recombinant expression of the antigen in organisms such as E.coli, yeast or mammalian cells would facilitate large scale production and thus benefit more humans.

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(hACE 2). This makes S-RBD a potential candidate as a subunit vaccine. However, the poor pharmacokinetics and low immunogenicity of S-RBD greatly hampers its development for subunit vaccines. One 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 scheme of the invention has the following advantages:

(1) the invention provides a nanogel capable of responding to reduction conditions and degrading to release S-RBD monomeric protein, and the immunogenicity of the S protein can be enhanced through the nanogel. The nanogel can improve lymph node targeting and the uptake of antigen presenting cells, and can be quickly converted into S-RBD monomer protein in the in-vivo immune process, so that a stronger immune response is generated.

(2) Under the condition of no adjuvant, the S-RBD nano gel provided by the invention can induce a quick and effective immune response by singly using the S-RBD nano gel, so that the nano gel has the prospect of being developed into a safer subunit vaccine.

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

(4) The invention provides a preparation method of S-RBD nanogel, which can produce S-RBD monomer protein in a large amount and safely in a heterologous expression mode and is used for the subsequent nanogel preparation. The preparation method has the advantages of simple steps, no pollution, good stability and the like.

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 only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1 is a schematic diagram illustrating the immune response elicited by the S-RBD nanogel provided by the invention.

FIG. 2 shows the results of SDS-PAGE and western blot analysis of the recombinantly expressed S-RBD protein.

FIG. 3 is a schematic diagram of the structure of an S-RBD nanogel;

a: reaction scheme for preparing nanogel by using S-RBD and a cross-linking agent;

b: schematic molecular structures of two crosslinking agents;

c: schematic representation of the breakdown of nanogels prepared from CL1 and CL2 in response to a reducing environment.

FIG. 4 shows the results of particle size analysis of S-RBD nanogels;

a: dynamic scattered light analysis (DLS) results for S-RBD nanogels;

b: transmission Electron Microscope (TEM) image of S-RBD nanogel; the scale bar shown in the figure is 10 nm.

FIG. 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 after 1h and 24 h; the scale bar shown in the figure is 50 μm.

FIG. 7 is a graph showing the analysis of S-RBD-NG uptake results by DC2.4 cells and RAW264.7 cells;

a: DC2.4 cells take up fluorescence confocal microscopy images of S-RBD-NG; the scale bar shown in the figure is 50 μm;

b: the quantitative analysis result of the S-RBD-NG uptake of DC2.4 cells;

c: the fluorescent confocal microscope image of S-RBD-NG taken up by RAW264.7 cells; the scale bar shown in the figure is 50 μm;

d: quantitative analysis of S-RBD-NG uptake by RAW264.7 cells.

FIG. 8 is a graph of experimental analysis of lymph node enrichment S-RBD-NG in mice;

a: the experimental process schematic diagram of the lymph node enrichment S-RBD-NG in the body of the mouse;

b: fluorescence imaging images of lymph nodes of a mouse body;

c: fluorescence quantitative analysis results of lymph nodes of the mouse body;

d: and analyzing the uptake of S-RBD-NG and S-RBD by DC cells and macrophages in the lymph nodes.

FIG. 9 shows the results of the antibody titer detection of mice after the second round of immunization;

a: detecting the detection result of S-RBD specific serum IgG in mouse serum by an ELISA method;

b: S-RBD specific serum IgG titre analysis plot calculated from A.

FIG. 10 shows the results of the antibody titer detection of the mice after the third round of immunization;

b: S-RBD specific serum IgG titre analysis plot calculated from A.

FIG. 11 shows the results of antibody titer measurements after immunization of mice with S-RBD-NG and Pam3CSK 4;

a: detecting the detection results of S-RBD specific serum IgG in the serum of the mice after the second round and the third round of immunization by an ELISA method;

b: S-RBD specific serum IgG titre analysis plot calculated from A.

FIG. 12 shows the results of detection of S-RBD interaction with hACE2 by competitive ELISA; wherein the horizontal axis represents the fold of serum dilution.

FIG. 13 shows the results of an experiment for neutralizing SARS-CoV-2 pseudovirus by an immune serum;

a: transfection inhibition of spike-PV-Luc by different titers of immune sera;

b: transfection inhibition of spike-PVGFP by immune sera.

FIG. 14 is a fluorescence confocal microscope image of the uptake of SARS-CoV-S1-NG by RAW264.7 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 to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1 construction of Pichia expression vector for S-RBD

Artificially synthesizing an encoding gene of a SARS-CoV-2S protein receptor binding domain (S-RBD) according to the codon preference of pichia pastoris, wherein the nucleotide sequence of the gene is SEQ ID NO: connecting the plasmid into a pPICZ alpha A vector by using an XhoI/NotI enzyme cutting site, converting the vector, selecting bleomycin resistant clones, extracting the plasmid to carry out PCR identification and sequencing identification, and identifying the correct recombinant plasmid, namely the recombinant expression plasmid pPICZ alpha A-S-RBD.

EXAMPLE 2 expression purification of S-RBD protein

1. Plasmid linearization

Taking 20 mu g of pPICZ alpha A-S-RBD plasmid, carrying out enzyme digestion on the pPICZ alpha A-S-RBD plasmid by using SacI in a water bath at 37 ℃, and identifying whether the pPICZ alpha A-S-RBD plasmid is linearized by using 1% agarose gel electrophoresis after enzyme digestion; after the linearization is determined, 1/10 volumes of sodium acetate and 2 volumes of absolute ethyl alcohol are added, and the mixture is inverted and mixed evenly; centrifuging at 14000rpm for 15min at 4 deg.C, and carefully removing the supernatant; the plasmid precipitate was washed with 500. mu.L of 75% ethanol, centrifuged at 14000rpm for 15min at 4 ℃, the supernatant carefully removed and air dried, and 30. mu.L of sterile water was added to resuspend the linearized plasmid.

2. Plasmid electrotransfer

Adding 10 mu L of linearized plasmid into 90 mu L X-33 yeast competence, mixing uniformly and adding into an electrode cup; putting the electrode cup into an electroporator, and carrying out electric shock at 2000V for 5 ms; adding 800 mu L of 1M sorbitol immediately after clicking, sucking all the sorbitol, transferring the sorbitol into a 15mL centrifuge tube, and performing shake culture for 2h at 25 ℃ and 200 rpm; then adding 3mL of non-resistance YPD culture medium, and carrying out shake cultivation for 2h at 25 ℃ and 200 rpm; centrifuging at room temperature 1100rpm, removing supernatant, resuspending the bacterial liquid with 300. mu.L YPD, adding 100. mu.L into YPD plate containing bleomycin; culturing in an inverted incubator at 28 deg.C for 2-3 days.

3. Positive clones Small batch screening

Individual clones were selected, added to 2.5mL YPD medium, and cultured at 28 ℃ with a shaker at 250rpm to OD ₆₀₀ 2-6, about 16-18 h; 150 μ L of BMGY medium was transferred to 3mL of BMGY medium with pH 6.5 and incubated at 28 ℃ with shaking at 250rpm to OD ₆₀₀ Is 1, about 8-12 h; replacing BMMY culture medium with methanol with final concentration of 0.5% to induce expression, adding methanol every 24h, and collecting supernatant after 72h to perform SDS-PAGE to identify positive clones.

4. Mass induced expression of positive clones

The positive clones selected in step 3 were added to 2.5mL YPD medium, cultured at 28 ℃ with shaking at 250rpm to OD ₆₀₀ Is 2-6, about 16-18 h; all of them were transferred to 100mL YPD medium and cultured at 28 ℃ and 250rpm on a shaker to OD ₆₀₀ 2-6, about 8-12 h; transferring 10mL of the culture medium into 200mL of BMGY medium, culturing at 28 ℃ and 250rpm to OD ₆₀₀ Changing to BMMY culture medium for heavy suspension for 1; adding methanol with final concentration of 0.5% to induce expression, wherein the methanol is added once every 24h, and the purification is carried out after 72 h.

5. Protein purification

Centrifuging the bacterial liquid obtained in the step (4) at 12000rpm for 20min, and taking the supernatant; the supernatant pH was adjusted to 8.0 using 1M Tris; centrifuging at 12000rpm, collecting supernatant, and filtering with filter paper; purifying the filtered supernatant by using an AKTA purification system and a Ni column; after purification, SDS-PAGE was performed to identify the protein, which was designated as S-RBD, as shown in FIG. 2, and SARS-CoV-2S protein receptor binding domain protein with a molecular weight of about 30kDa was obtained after purification.

Example 3 chemical crosslinking of S-RBD protein monomers into multimers

The S-RBD protein monomer purified in example 2 is cross-linked into multimer by cross-linking agent, and the schematic structure of multimer is shown in A in FIG. 3. The method comprises the following specific steps:

the S-RBD protein monomer prepared in example 2 is mixed with a cross-linking agent CL1 shown in formula i or a cross-linking agent CL2 shown in formula ii with molar equivalents of 10, 20 and 50 respectively, and incubated for 1h at 30 ℃ with continuous shaking. The reaction mixture was then passed through a PD-10 column to remove excess crosslinker;

two nanogels with different spacer groups were prepared, the schematic of the spacer groups is shown as C in fig. 3, and both contain a disulfide bond inside. After the nanogel is taken up by Antigen Presenting Cells (APCs), disulfide bonds are reduced, and both the nanogel prepared from CL1 (designated as S-RBD-CL1) and the nanogel prepared from CL2 (designated as S-RBD-CL2) can be decomposed to release S-RBD protein monomers. As shown in FIG. 3C, the protein monomer reduced by S-RBD-CL1 has thiol groups, and the protein monomer reduced by S-RBD-CL2 can restore the natural amino groups.

Dynamic scattering optical analysis (DLS) and Transmission Electron Microscope (TEM) imaging were performed on 50 molar equivalents of the nanogel prepared with CL2, the results of which are shown in fig. 4 a and fig. 4B. According to this measurement, the average diameter of the crosslinked nanogels is about 25nm, whereas the diameter of the native S-RBD is about 2nm as measured by DLS.

Example 4 monomeric and multimeric labelling of Cy5.5 fluorescence

The monomer obtained in the example 2 and the polymer obtained in the example 3 are respectively added into Tris/HCl buffer solution with pH9.0, after fully mixing, Cy5.5-NHS is added into the mixture, the mixture is quickly mixed, the mixture is put into a mixer with 25 ℃ and 1000rpm for reaction overnight (whole process is protected from light), the unreacted Cy5.5-NHS is removed by a desalting column, finally, the mixture is filtered by a 0.22 mu M filter membrane, and the products after reaction are respectively marked as S-RBD-Cy5.5, S-RBD-CL1-Cy5.5, S-RBD-CL2-Cy5.5, and are stored in the condition of being protected from light at 4 ℃.

Example 5SDS-PAGE identification

The S-RBD-CL1-Cy5.5 with different equivalent CL1 and the S-RBD-CL2-Cy5.5 with different equivalent CL2 prepared in the example 4 are respectively mixed with equal volume of 2 Xdenaturation loading buffer solution and non-denaturation loading buffer solution, the mixture is boiled at 100 ℃ for 10min, after natural cooling, the mixture is 12000rpm and centrifuged for 1min, and 30 mu L of the mixture is loaded into a gel loading hole. The concentration of the separation gel is 12%, and electrophoresis is carried out for 1h at constant voltage of 140V. After electrophoresis, the bands were stained with Coomassie Brilliant blue and visualized by Cy5.5 fluorescence.

The results are shown in FIG. 5, where the denatured band shows a monomeric size with a molecular mass of about 30kDa and the non-denatured band is multimeric in nature with a size of about 22 kDa. The results indicate that the nanogel formed from CL2 showed higher efficiency.

Example 6 uptake by antigen presenting cells

The uptake of antigen by antigen presenting cells is critical to antigen processing and cross presentation, and this example demonstrates the uptake capacity of antigen presenting cells for S-RBD-CL1 and S-RBD-CL 2; and further quantitatively analyzing the uptake capacity of the antigen presenting cells to nanogels with different ratios of the cross-linking agent and the S-RBD. The method comprises the following specific steps:

DC2.4 cells were treated with S-RBD-Cy5.5(0.1nmol) prepared in example 4, while DC2.4 cells were treated with S-RBD-CL1-Cy5.5(0.1nmol) of CL1 in different equivalents and S-RBD-CL2-Cy5.5(0.1nmol) of CL2 in different equivalents, respectively, as prepared in example 4, and after the completion of co-incubation, they were washed three times with PBS, and then DC2.4 cells were stained by adding Hoechst (staining nuclei), and then DC2.4 surface fluorescence was observed with a confocal microscope.

As shown in FIG. 6, S-RBD nanogels prepared using CL1 and CL2 both aggregated significantly more in DC2.4 cells than S-RBD monomer. Since it is considered that the nanogel prepared from CL2 can restore the natural S-RBD protein by reduction, S-RBD nanogel (named S-RBD-NG and used hereinafter) formed from CL2 is preferable for the next study and experiment.

When DC2.4 cells and RAW264.7 cells were treated with S-RBD-NG (0.1nmol) at different cross-linker/S-RBD molar ratios (10X, 20X, 50X) according to the above method, the results of fluorescence observation are shown in FIGS. 7A and 7C, and the results of quantitative analysis of imaging data are shown in FIGS. 7B and 7D, showing that the uptake effect of S-RBD-NG is affected by CL2 equivalent and that nanogel effect is optimal when 50 molar equivalent CL2 is used and that the uptake of antigen presenting cells can be increased by about 4 times, compared with S-RBD monomer.

Example 7S-RBD-NG increases lymph node enrichment

Injecting 0.66nmol S-RBD-Cy5.5, 10 molar equivalent CL 2S-RBD-NG-Cy5.5, 50 molar equivalent CL 2S-RBD-NG-Cy5.5 and equivalent Cy5.5 into C57BL/6N mice muscle respectively, killing the mice by cervical dislocation after 24h injection as shown in A in figure 8, spraying 75% alcohol on the surface, fixing the limbs of the mice on an dissecting table by using a pin, cutting the skin of the mice by using scissors, peeling the skin, searching the inguinal lymph node of the mice, taking down the lymph node by using tweezers, imaging the lymph node by using a Maestro mouse imaging system, wherein the imaging picture of the lymph node is shown in B in figure 8, and the result of fluorescence quantitative analysis is shown in C in figure 8;

from B in FIG. 8, it can be seen that S-RBD-NG-Cy5.5 has higher aggregation and longer retention time in mouse lymph node than S-RBD-Cy5.5, indicating that the multimer prepared by the present invention can significantly increase lymph node enrichment. Further quantitative analysis (C in FIG. 8) indicated that the accumulation of S-RBD-NG was increased by about 3.9 times for 50 molar equivalents of CL2 compared to S-RBD.

After digestion of lymph nodes into single cells, the uptake of S-RBD-NG and S-RBDCY5 by DC cells and macrophages was analyzed by flow cytometry. As a result, as shown in D in FIG. 8, it is found that the uptake of S-RBD-NG by DC cells and macrophages is significantly higher than that by S-RBD.

Example 8 immunogenicity testing

This example tests S-RBD-NG for immunogenicity in vivo. The method comprises the following specific steps:

c57BL/6N mice were immunized intramuscularly with the following reagents, respectively: PBS, S-RBD (50. mu.g/mouse), S-RBD + aluminum adjuvant (50. mu.g/mouse of S-RBD, 100. mu.g/mouse of aluminum hydroxide), S-RBD-NG (50. mu.g/mouse), S-RBD-NG + aluminum adjuvant (50. mu.g/mouse of S-RBD-NG, 100. mu.g/mouse of aluminum hydroxide).

Phase was used on day 14 and day 28 of first immunizationMice were further boosted at the same dose and sera were collected one week after each immunization (i.e., 7, 21, 35 days after the first immunization). S-RBD specific serum IgG was detected by enzyme-linked immunosorbent assay (ELISA) and titer was calculated. One week after the first immunization, IgG titers in all groups remained below the limit of detection (below the lowest dilution factor of 50, data not shown). After the second round of immunization, the S-RBD-NG treatment group increased serum IgG titers to-10 in the presence and absence of aluminum adjuvant ⁴ (A in FIG. 9 and B in FIG. 9). After the third immunization round, the titer of the S-RBD-NG treatment group reached-10 ⁵ And the titer of the S-RBD monomer treatment group is less than 10 ⁴ (A in FIG. 10 and B in FIG. 10). Quantitative analysis shows that S-RBD-NG induces 27.6 times higher potency than S-RBD monomer (without aluminum adjuvant) and 8.3 times higher potency than S-RBD monomer (with aluminum adjuvant); S-RBD-NG is more immunogenic than S-RBD and can elicit a more effective and rapid immune response.

Example 9 enhancement of immunogenicity with Pam3CSK4 as adjuvant

Aluminum hydroxide is one of the most commonly used adjuvants in the art, however, it has no significant improvement effect on the immunogenicity of S-RBD-NG according to the results of example 8. The invention explores a plurality of adjuvants and finds that the immune potency of the S-RBD-NG can be obviously improved when Pam3CSK4 is used as the adjuvant of the S-RBD-NG.

This example tests the toll-like receptor 1/2 agonist Pam3CSK4 (5 nmol per injection) in combination with S-RBD-NG to elicit a more potent immune response, as described in example 8, see A in FIG. 11 and B in FIG. 11, and S-RBD-NG specific IgG titers of-10 after the third immunization round ⁶ . This indicates that the immune titer of S-RBD-NG can be further improved by optimizing the use of adjuvants.

Example 10 production of specific antibodies induced by S-RBD-NG

Since blocking the interaction between spike protein and ACE2 is important to prevent SARS-CoV-2 from entering the host cell, this example tested whether serum from S-RBD-NG immunized mice could inhibit this interaction. The method comprises the following specific steps:

after immunization of the mice in examples 8-9, serum collection was performed: blood was collected via orbital vein 1 week after each immunization, and the blood samples were placed in EP tubes at room temperature for 1h, then centrifuged at 4000rpm at room temperature for 10min, and the supernatants were collected as serum samples.

Competition ELISA: S-RBD was diluted to 1. mu.g/mL with PBS, plated onto EIA plates at 4 ℃ and allowed to stand overnight. Wash once with PBST (0.5% tween-20). Then blocked with 2% BSA in PBS for 2 h. The serum was diluted with PBST-BSA (0.5% Tween, 0.5% BSA), pre-blocked with 50. mu.L of different dilutions of serum for 30min (with serum-free blocking controls), added with 50. mu.LACE-hFc (1. mu.g/mL) and incubated for an additional 1 h. 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 at room temperature for 1 h. Then, the reaction was stopped by washing 4 times with PBST, adding 100 μ L of TMB to each well, and after incubation at room temperature, adding 50 μ L H2SO4(2N) to each well. The absorbance at 450nm was immediately measured. The detection results are shown in fig. 12.

As can be seen in FIG. 12, S-RBD-NG immunized sera effectively blocked the interaction of S-RBD with hACE 2. This indicates that the S-RBD-NG prepared by the invention induces specific antibodies in vivo, and can target and block the interaction between S-RBD and hACE 2.

Example 11 neutralization of SARS-CoV-2 pseudovirus by S-RBD-NG

This example used the sera of immunized mice obtained in examples 8-9 to neutralize the SARS-CoV-2 pseudovirus to test the effectiveness of S-RBD-NG as a pre-antigen for use in a SARS-CoV-2 subunit vaccine. The SARS-CoV-2 pseudovirus used in this example had a spike protein shell and carried a luciferase gene (called spike-PV-Luc) as a reporter gene. The method comprises the following specific steps:

COS7-hACE2 cells (COS 7 cell line stably expressing hACE2) were seeded in a 96-well plate at a ratio of 1:30 and cultured for 24 hours. spike-PV-Luc pseudoviruses were then incubated with different dilutions (dilutions 1:20, 1:40) of serum for 1h on ice. Then, the pseudovirus and serum mixture was added to COS7-hACE2 cells, and the medium was replaced with fresh medium after 24 hours of culture, followed by further 24 hours of culture. After the culture was completed, the cells were collected and lysed, and the transfection efficiency was measured by detecting the fluorescence intensity using a luciferase reporter.

By examining the transfection efficiency of spike-PV-Luc pseudovirus, the neutralizing activity of serum can be evaluated. As can be seen from the results in FIG. 13, the sera from PBS and S-RBD immunized mice had no significant inhibition; serum of S-RBD-NG immunized mice can obviously inhibit the transfection efficiency of pseudoviruses and is concentration-dependent; whether the aluminum adjuvant is added or not is not obviously different in the experiment; serum from mice immunized with S-RBD-NG and Pam3CSK4 almost completely inhibited pseudovirus transfection at both dilution times.

From the results A in FIG. 13, it was found that the transfection efficiency of pseudovirus was enhanced when serum was used at a higher concentration (1:20 dilution) than when serum was used at a lower concentration (1:40 dilution). This is probably due to the fact that SARS-CoV-2 can promote viral entry (i.e., antibody-dependent enhancement) upon infection with antibodies in the blood. To further confirm this result, another spike pseudovirus carrying the gfp gene (called spike-PVGFP) was prepared in this example. The efficiency of Spike PV-GFP transfected COS7-hACE2 cells was tested by serum diluted 1:20 as described in the previous experimental procedures, and the results of confocal microscopy are shown in FIG. 13, panel B. The results in FIG. 13, B, show that sera from mice immunized with S-RBD-NG were effective in neutralizing the SARS-CoV-2 pseudovirus.

Example 12

Using a method similar to examples 1-7, this example prepared recombinant S1 subunit of SARS-CoV-1 and CL2 into a multimeric nanogel (named SARS-CoV-S1-NG) and verified the uptake of raw264.7 cells. As shown in FIG. 14, when SARS-CoV-S1 was prepared as a nanogel, intracellular uptake was significantly increased as compared with that of S1 protein monomer. This shows that other coronaviruses or other viruses infecting cells with glycoprotein-binding cell receptors, such as ebola virus, respiratory syncytial virus, etc., can be used to develop subunit vaccines by preparing the receptor-binding domain of the glycoprotein into the nanogels of the invention to enhance the uptake by antigen presenting cells, induce a more rapid and effective immune response, and based thereon.

While the invention has been described with reference to specific preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

<110> Beijing university

<120> spike protein receptor binding structural domain nanogel and preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 223

<212> PRT

<213> SARS-CoV-2

<400> 1

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 2

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agagtacaac caactgaatc cattgttaga tttcctaata tcactaacct gtgcccattt 60

ggtgaagttt ttaacgctac tagatttgct tctgtttacg cctggaacag aaagagaatt 120

tctaactgtg ttgctgatta ctctgttctt tacaactctg cctctttttc tacttttaag 180

tgttatggtg tctctccaac caagttgaac gatttgtgtt ttaccaacgt ttacgctgat 240

tcttttgtta ttagaggtga tgaggttaga caaattgctc ctggtcaaac tggtaagatt 300

gctgattata actacaagtt gcctgatgat tttactggtt gcgtcattgc ttggaactct 360

aataatttgg attctaaggt tggtggaaat tacaactact tgtacagatt gtttagaaag 420

agtaacttga agccatttga aagagatatt tctactgaaa tctaccaagc tggatctact 480

ccttgtaacg gtgtcgaagg ttttaactgc tactttcctt tgcagtctta cggttttcaa 540

cccactaacg gtgttggtta ccagccctac agagttgttg ttttgtcttt tgagttgctt 600

catgctccag ctactgtttg tggtcctaag aagtctacta acttggttaa gaacaagtgt 660

gttaatttc 669

Claims

1. A nanogel is characterized in that the nanogel is obtained by chemically crosslinking a SARS-CoV-2 virus spike protein receptor binding domain and a crosslinking molecule; the amino acid sequence of the SARS-CoV-2 virus spike protein receptor binding structure domain is SEQ ID NO: 1, the structural formula of the cross-linking molecule is shown as formula I:

wherein the content of the first and second substances,

R ₁ and R ₂ Each independently selected from-CH ₂ -or-O-;

n and m are each independently selected from integers of 1 to 5.

2. The nanogel of claim 1 wherein said cross-linking molecule has a structural formula according to formula i or formula ii:

3. the nanogel of claim 1 or claim 2, wherein the molar ratio of said cross-linking molecule to said SARS-CoV-2 viral spike protein receptor binding domain is 10 to 50: 1.

4. The nanogel of claim 1 or claim 2 wherein the nanogel has a particle size of 16 to 50 nm.

5. A process for the preparation of a nanogel as claimed in any one of claims 1 to 4 comprising the steps of: and mixing the SARS-CoV-2 virus spike protein receptor binding structural domain with the cross-linking molecule, incubating and purifying to obtain the nanogel.

6. The method according to claim 5, wherein the incubation temperature is 20 to 35 ℃; the incubation time is 0.5-2 h.

7. The method of claim 5, wherein the purifying comprises passing through a PD-10 column to remove excess cross-linking molecules.

8. The method of claim 5, wherein the SARS-CoV-2 viral spike protein receptor binding domain is heterologous expressed.

9. The method of claim 8, wherein said heterologous expression comprises the steps of: obtaining coding gene of SARS-CoV-2 virus spike protein receptor binding structure domain; constructing a host cell capable of expressing the encoding gene; culturing the host cell under culture conditions suitable for expression of the SARS-CoV-2 viral spike protein receptor binding domain; collecting the culture product and separating and purifying the SARS-CoV-2 virus spike protein receptor binding structural domain.

10. The method of claim 9, wherein the host cell is an escherichia coli, yeast, or mammalian cell.

11. The method of claim 9, wherein the encoding gene is codon-biased for the host cell.

12. The method of claim 9, wherein the nucleotide sequence of the coding gene is SEQ ID NO: 2.

13. use of a nanogel according to any of claims 1 to 4 in:

(1) preparing a vaccine of SARS-CoV-2 virus;

(2) preparing the immune enhancing medicine of SARS-CoV-2 virus.

14. The use of claim 13, wherein the vaccine is a subunit vaccine.

15. A vaccine composition comprising the nanogel of any one of claims 1 to 4 and an acceptable vaccine adjuvant.

16. The vaccine composition of claim 15, wherein the vaccine adjuvant is Pam3CSK 4.