CN112043825A

CN112043825A - Subunit vaccine for preventing novel coronavirus infection based on novel coronavirus spike protein S1 region

Info

Publication number: CN112043825A
Application number: CN202010867895.4A
Authority: CN
Inventors: 刘存宝; 曹晗; 王云飞; 王丽春
Original assignee: Institute of Medical Biology of CAMS and PUMC
Current assignee: Institute of Medical Biology of CAMS and PUMC
Priority date: 2020-07-13
Filing date: 2020-08-26
Publication date: 2020-12-08
Anticipated expiration: 2040-08-26
Also published as: CN112043825B

Abstract

The invention provides a subunit vaccine for preventing novel coronavirus infection based on a novel coronavirus spike protein S1 region, and belongs to the field of vaccines. The subunit vaccine comprises the novel coronavirus spike protein S1 region antigen and adjuvant, and is administered by subcutaneous or intramuscular injection for 2-3 times for preventing novel coronavirus. The subunit vaccine of the invention can avoid the potential ADE risk of the full-length S protein serving as a novel coronavirus vaccine, retain the immunogenicity of RBD but not introduce other virus antigens, and ensure that the antibody generated after immunization can sufficiently neutralize the novel coronavirus.

Description

Subunit vaccine for preventing novel coronavirus infection based on novel coronavirus spike protein S1 region

Technical Field

The invention belongs to the field of vaccines, and particularly relates to a subunit vaccine for preventing novel coronavirus infection based on a novel coronavirus spike protein S1 region.

Background

The new coronavirus (SARS-CoV-2) found at the end of 12 months in 2019 is worldwide half a year, causing infection of more than ten million people and death of more than 50 million people by 6 months in 2020. Clinically used drugs including clindamycin (lopinavir/ritonavir), hydroxychloroquine, reicepivir and dexamethasone for the emergency treatment of neocoronary pneumonia (coronavirus disease 2019, COVID-19) did not show satisfactory results. In view of the strong infectivity and high pathogenic, lethal rate of SARS-CoV-2, the vaccine remains the first choice to cope with COVID-19 in addition to public defense.

A variety of vaccine formats, including nucleic acid vaccines (DNA/RNA), viral vector vaccines, attenuated/inactivated vaccines and subunit vaccines are being evaluated clinically. Attenuated/inactivated vaccines and subunit vaccines are common forms of marketed vaccines (nucleic acid vaccines do not currently have any products on the market, and viral vector vaccines only have vaccines against ebola virus on the market), wherein the development cycle of attenuated vaccines is not long enough to meet the urgent need for vaccines at present; the exposure risk caused by the strong infectivity of the virus in the production process of the inactivated vaccine causes that the requirements on factory conditions and production regulations are higher. In addition, based on previous experience in developing coronavirus vaccines (including SARS-CoV and middle east respiratory syndrome virus MERS-CoV), inactivated vaccines have resulted in antibody-dependent enhanced infection (ADE) in animal experiments, and this risk has led to the withdrawal of clinical phase 1 experiments for American SARS inactivated vaccine (NCT 00533741).

The Spike protein (Spike, S) of coronavirus is responsible for the binding of virus and host cell receptor (region S1) and mediating the membrane fusion of virus and host cell (region S2), and the S protein antibody titer is closely related to the severity and survival rate of infected patients, so that the Spike protein (S) is the first choice antigen for the development of coronavirus vaccine. Unfortunately, the combination of the full-length protein S and the aluminum adjuvant has a certain protective effect on virus attack after immunization, but severe lung immunopathology characterized by eosinophil infiltration is induced by virus infection. This Th 2-type immune response presents a fatal risk to vaccinees with basic respiratory diseases such as asthma, lung obstruction, etc., such as many elderly people, which has led to the withdrawal of the us S protein-based SARS subunit vaccine phase 1 clinical trial (NCT 01376765).

The use of only the Receptor Binding Domain (RBD) of coronaviruses as a subunit vaccine is believed to be effective in reducing the risk of ADE, but due to its small molecular weight (about 200 amino acids), it is desirable to display it on the surface of virus-like particles of other viruses (which introduce non-essential antigens) or to prepare it in multimeric form to enhance immunogenicity. Therefore, an antigen selection that both reduces the risk of ADE and is sufficiently immunogenic is of great importance.

Disclosure of Invention

In view of the above problems, the present invention provides a subunit vaccine for preventing infection by a novel coronavirus based on the region of novel coronavirus spike protein S1, which can avoid the potential ADE risk of using the full-length S protein as a novel coronavirus vaccine, and can retain the immunogenicity of RBD without introducing other viral antigens, so that the antibodies generated after immunization can sufficiently neutralize the novel coronavirus.

The purpose of the invention is realized by the following technical scheme:

a subunit vaccine for preventing a novel coronavirus infection based on a novel coronavirus spike protein S1 region, wherein the subunit vaccine comprises a novel coronavirus spike protein S1 region antigen and the amino acid sequence of the subunit vaccine is as follows:

VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR。

further, the subunit vaccine also comprises an adjuvant.

Further, the novel coronavirus spike protein S1 region antigen is a novel coronavirus spike protein S1 region prepared by expression of mammalian cells, an insect-baculovirus system and a yeast system.

Further, in the single-needle vaccine, the content of the antigen of the S1 region is 5 to 500. mu.g.

Further, the adjuvant comprises an aluminum hydroxide adjuvant, and in the single-needle vaccine, the content of the adjuvant is 0.3-3 mg.

Further, the subunit vaccine is administered 2-3 times by subcutaneous or intramuscular injection for the prevention of novel coronavirus infection.

Another aspect of the invention:

the application of the subunit vaccine for preventing the novel coronavirus infection based on the novel coronavirus spike protein S1 region in preventing the novel coronavirus infection is disclosed.

Compared with the prior art, the invention has the beneficial effects that:

(1) the present invention avoids the risk of ADE as much as possible by removing the membrane fusion region S2 of the S protein mediating virus and host cell; the RBD in the receptor binding region of virus and host cells is reserved in the S protein, and simultaneously, the N-terminal functional region (NTD) in the S1 region is reserved so as to maintain the spatial conformation of the RBD and the immunogenicity related to the RBD;

(2) experiments prove that the serum antibody generated by the S1 protein immune experimental animal can effectively neutralize the novel coronavirus; the IgG2a titer of the antibodies generated by S1 protein immunization experimental animals is higher, and the ADE risk is lower.

Drawings

FIG. 1 shows the protein electrophoretogram (A), the immunoblot identification result (B) and the transmission electron microscopy detection (C and D) of Nor core protein-novel coronavirus spike protein S1 (S-S1), Nor core protein-novel coronavirus receptor binding domain protein RBD (S-RBD) prepared in example 1 of the present invention;

FIG. 2 shows IgG (B) and IgA (C) antibody titers specific for the S1 antigen and RBD antigen measured by example 3 after 3 intramuscular injections for 2 weeks at 2-week intervals of example 2;

FIG. 3 shows IgG1(A) and Ig2a (B) antibody titers specific for the S1 antigen and RBD antigen measured by example 3 after 2 weeks of 3 intramuscular injections 2 weeks apart by example 2, and the ratio of the IgG1 to Ig2a calculated by example 4;

figure 4 shows the neutralizing antibody titers of the novel coronavirus measured by examples 5 and 6 after 2 weeks of 3 intramuscular injections at 2 weeks intervals in example 2.

Detailed Description

Example 1 preparation of antigen

The genes encoding the S1 (YP _009724390.1, Met1-Tyr 695) and RBD (YP _009724390.1, Arg328-Pro 521) regions of the novel coronavirus were linked to the gene encoding the core protein of norovirus, synthesized by Nanjing Kirsii after optimization according to the codon preference of Escherichia coli, constructed between the BamHI and NotI cleavage sites of plasmid pET-28b, and then subjected to sequencing and identification, transformed into Escherichia coli (BL-21, DE 3), and 0.4 mM isopropyl-beta-D-thiogalactoside was induced overnight at room temperature (. about.25 ℃). Centrifuging at 8000 rpm at 4 deg.C for 15 min to collect thallus, resuspending in PBS solution, ultrasonically washing at 8000 rpm at 4 deg.C for 15 min, centrifuging at 8000 rpm at 4 deg.C for 60 min to collect precipitate, ultrasonically washing again for 15 min, centrifuging at 8000 rpm at 4 deg.C for 15 min to collect inclusion body, dissolving in 8M urea, and dialyzing at 4 deg.C with 20 mM Tris-HCl (pH 7.4) solution containing 6M, 4M, 2M, 1M and 0M. The prepared protein was determined in concentration using a TCA protein concentration detection kit (bi yun day), protein purity was detected by electrophoresis using 10% SDS-PAGE protein, identified using anti-SARS-CoV-1S 1 polyclonal antibody (yokekan, seiko) and goat anti-rabbit polyclonal antibody (semefying) labeled with horseradish peroxidase, and formation of virus-like particles was detected using transmission electron microscopy (hiti).

Results referring to FIG. 1, the results of protein electrophoresis and immunoblot identification showed that both RBD (S-RBD, about 52 kDa) and S1 (S-S1, about 108 kDa) fused to norovirus core protein were efficiently expressed (FIG. 1B). The purity of S-S1 was approximately 60% relative to high purity S-RBD (FIG. 1A). A small fraction of both proteins formed virus-like particles with diameters of about 30-60 nm, but the vast majority of proteins appeared to be irregularly aggregated (FIGS. 1C and 1D).

Example 2 animal immunization

Mammalian cell HEK293 expressed S1 and RBD proteins (see, kyo), as well as e.coli expressed S-S1 and S-RBD proteins prepared in example 1 were diluted to 10 μ g/25 μ L, mixed with the same volume of aluminum hydroxide adjuvant (40 mg/mL), and then immunized with BALB/c mice (6 weeks old, 14-17 g, purchased from institute of medicine and biology, experimental animals center, chinese academy of medicine) by 3 intramuscular injections 2 weeks apart. After 2 weeks of final immunization the heart was bled and placed overnight at 4 ℃ and then centrifuged at 3000 rpm for 10 minutes to obtain serum ready for subsequent immunological analysis.

Wherein, the amino acid sequence of the S1 protein is as follows:

example 2 the procedure is shown in figure 2A.

Example 3 antibody Titer assay

Adding 100 mu L of S1 and RBD protein 2 mu g/mL of HEK293 expression of antigen-capturing mammal cells dissolved in PBS into a 96-well enzyme-linked immunosorbent assay plate (Corning), washing the plate 1 time by TBST (0.05% Tween20 (Sigma) in PBS) after overnight coating at 4 ℃, adding 5% (w/v) of skimmed milk powder dissolved in PBS into 200 mu L of each well at 37 ℃ for sealing for 1 h, washing the TBST 4 times after discarding the milk, adding 1% of antiserum prepared in example 2 diluted by a milk gradient into 100 mu L of each well at 37 ℃ for incubating for 1 h, adding 1% of horse radish peroxidase-labeled secondary goat anti-mouse IgG/IgA/IgG1/IgG2a (Xemic fly) diluted by milk after washing for 5 times by TBST, adding 100 mu L of light-shielding solution (purchased from BD) prepared according to the ratio of 1:1 to each well after washing for 5 times by TBST, and placing the plate for 10 minutes at room temperature, 100 μ L of 2M sulfuric acid was added to each well to terminate the reaction, and the light absorption value was measured at 450 nm. The antibody titer was determined as the concentration of a critical serum dilution with OD450 < 0.1.

The results of the antibody titer test are shown in FIG. 2. Immunization of mammalian cells S1 expressed by HEK293 and S-S1 expressed by E.coli induced the same titers of S1-specific IgG antibodies (64000, FIG. 2B), S1-specific IgA antibodies (64000, FIG. 2C) and RBD-specific IgG antibodies (8000, FIG. 2B), and the lack of enhancement of the S1 protein by norovirus core protein was most likely due to its only 60% purity. Coli expressed S-RBD induced higher titers of all antibody types relative to mammalian cell HEK293 expressed RBD (fig. 2B and 2C). It is noteworthy that although S-RBD induced the highest concentrations of IgG and IgA antibody specific to RBD, it induced lower titers of both IgG and IgA antibodies specific to S1 than the S1 group. In conclusion, the immunogenicity of the S1 protein is much higher than that of the RBD.

FIG. 4 shows IgG1(A) and Ig2a (B) antibody titers specific for S1 antigen and RBD antigen.

Example 4 ADE Risk assessment

According to the IgG1/IgG2a antibody titer ratio obtained in example 3, the Th1/Th2 response condition caused by the immune antigen is evaluated through calculation. The results are shown in FIG. 4.

Based on the IgG1 (FIG. 3A) and IgG2a (FIG. 3B) antibody titers measured in example 3, it was found from example 4 that the IgG2a antibody titer obtained after immunization with S1 protein was much higher than the IgG2a antibody titer obtained after immunization with RBD, showing that S1 protein was more inclined to Th1 response than RBD after immunization as antigen, with lower risk of inducing antibody-enhanced infection.

Example 5 Virus Titer confirmation

100 μ L Vero cells were suspended in DMEM medium (Corning) containing 5% fetal bovine serum (Hyclone), 2.5X 10⁵After being inoculated in a 96-well plate, the plate is placed in a chamber containing 5% CO₂The incubator of (1) was incubated at 37 ℃ overnight. The novel coronavirus strain KMS-1 (GenBank No: MT 226610.1) was diluted 10-fold with DMEM and added to the cells at 100. mu.L/well. 5% CO₂The incubator is cultured at 37 ℃ for 6-7 days, and then the cytopathic effect is observed by an inverted microscope. The dilution factor of the virus that caused 50% of the cytopathic effects was used to calculate the half infection in cell culture (CCID 50).

Example 6 Virus neutralization Titer assay

The immune serum was diluted with DMEM at a double ratio, 50. mu.L was incubated with KMS diluted with DMEM at 3.3lg CCID50 for 1 hour at 100. mu.L/well containing 2.5X 10⁵10% fetal bovine serum DMEM medium of vero cells, and the dilution factor of antiserum which inhibited infection by 50% of cells was recorded as the virus-neutralizing antibody titer after 4 days of culture at 37 ℃.

The results of the test are shown in FIG. 4. The results show that only S1 expressed by the HEK293 of the mammalian cells induces obvious virus neutralizing antibody titer, and further confirm that the S1 protein has better immune effect compared with RBD. Notably, while S1 expressed by mammalian cell HEK293 and S-S1 expressed by e.coli induced the same titers of S1-specific IgG antibodies (64000, fig. 2B), S1-specific IgA antibodies (64000, fig. 2C) and RBD-specific IgG antibodies (8000, fig. 2B), S-S1 did not induce significant virus-neutralizing antibodies as did the S1 protein. The result indicates that the final immune protection effect of the S1 protein can be greatly influenced by post-translational modification or spatial structure change of the protein, and mammalian cells and other expression systems with higher fidelity are preferentially selected in the process of preparing the S1 protein as a vaccine antigen.

<110> institute of medical science and biology of China academy of medical sciences

<120> a subunit vaccine for preventing novel coronavirus infection based on novel coronavirus spike protein S1 region

<140> 202010867895.4

<151> 2020-07-13

<160> 1

<210> 1

<211> 670

<212> PRT

<213> Artificial sequence

<400> 1

1 VNLTTRTQLP PAYTNSFTRG VYYPDKVFRS SVLHSTQDLF LPFFSNVTWF HAIHVSGTNG

61 TKRFDNPVLP FNDGVYFAST EKSNIIRGWI FGTTLDSKTQ SLLIVNNATN VVIKVCEFQF

121 CNDPFLGVYY HKNNKSWMES EFRVYSSANN CTFEYVSQPF LMDLEGKQGN FKNLREFVFK

181 NIDGYFKIYS KHTPINLVRD LPQGFSALEP LVDLPIGINI TRFQTLLALH RSYLTPGDSS

241 SGWTAGAAAY YVGYLQPRTF LLKYNENGTI TDAVDCALDP LSETKCTLKS FTVEKGIYQT

301 SNFRVQPTES IVRFPNITNL CPFGEVFNAT RFASVYAWNR KRISNCVADY SVLYNSASFS

361 TFKCYGVSPT KLNDLCFTNV YADSFVIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA

421 WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPCNGVEG FNCYFPLQSY

481 GFQPTNGVGY QPYRVVVLSF ELLHAPATVC GPKKSTNLVK NKCVNFNFNG LTGTGVLTES

541 NKKFLPFQQF GRDIADTTDA VRDPQTLEIL DITPCSFGGV SVITPGTNTS NQVAVLYQDV

601 NCTEVPVAIH ADQLTPTWRV YSTGSNVFQT RAGCLIGAEH VNNSYECDIP IGAGICASYQ

661 TQTNSPRRAR

Claims

1. A subunit vaccine for preventing a novel coronavirus infection based on the S1 region of the novel coronavirus spike protein, wherein the subunit vaccine comprises the S1 region antigen of the novel coronavirus spike protein and has the following amino acid sequence:

2. the subunit vaccine of claim 1, further comprising an adjuvant.

3. The subunit vaccine of claim 1, wherein the novel coronavirus spike protein S1 domain antigen is a novel coronavirus spike protein S1 domain prepared by expression using mammalian cells, insect-baculovirus systems, yeast systems.

4. The subunit vaccine of claim 1, wherein the amount of the S1 domain antigen is 5 μ g to 500 μ g in a single needle vaccine.

5. The subunit vaccine of claim 2, wherein the adjuvant comprises an aluminium hydroxide adjuvant, and wherein the adjuvant is present in an amount of 0.3 to 3 mg in the single needle vaccine.

6. The subunit vaccine of any one of claims 1 to 5, wherein the subunit vaccine is administered subcutaneously or intramuscularly.

7. Use of a subunit vaccine according to any of claims 1 to 6 for the prophylaxis of novel coronaviruses.