WO2022253134A1 - Method for improving immunogenicity/antigenic trimer stability of ecd antigen of sars-cov-2 mutant strain - Google Patents

Abstract

The present invention relates to the field of molecular vaccinology, and provides a method for improving the immunogenicity/antigenic trimer stability of an extracellular domain (ECD) antigen of a SARS-CoV-2 mutant strain, and an ECD immunogenic protein/peptide, having improved immunogenicity/antigenic trimer stability, of the SARS-CoV-2 mutant strain. The present invention comprises, but is not limited to, an ECD of a spike protein (S protein) of a SARS-CoV-2 strain, a B.1 strain, a B.1.1.7 strain or a B.1.351 strain having a genome sequence number of GenBank Accession No. MN908947.3; by introducing a homotrimer formed by a mutation site and a trimerization-assisted structure, the immunogenicity/antigenic trimer stability of the ECD antigen is improved. A vaccine further comprises a pharmaceutically acceptable adjuvant. A vaccine composition exhibits excellent immunogenicity in mice and Macaca fascicularis, and can maintain long-term humoral and cellular immune responses. A recombinant trimer protein vaccine can be used for preventing diseases related to SARS-CoV-2 infections.

Description

A method for improving the immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strain ECD antigen

Cross References to Related Applications

This application claims the benefit of Chinese patent application 202110606512.2 filed on May 31, 2021 and Chinese patent application 202111237604.4 filed on October 22, 2021, the contents of which are incorporated herein by reference.

technical field

The invention relates to the field of molecular vaccinology, and relates to a method for improving the ECD antigen immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strains and a SARS-CoV-2 improved immunogenicity/antigen trimer stability - CoV-2 mutant strain ECD immunogenic proteins/peptides.

Background technique

The new coronavirus (SARS-CoV-2) has a strong ability to spread, and a safe and effective vaccine is the most powerful technical means to control the epidemic. According to different targets and technologies, vaccines can be divided into the following categories: inactivated vaccines, recombinant protein vaccines, viral vector vaccines, RNA vaccines, live attenuated vaccines, and virus-like particle vaccines.

The trimeric Spike protein (Spike, S protein) of SARS-CoV-2 is the main component of the virus envelope and plays an important role in receptor binding, fusion, virus entry and host immune defense. SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin-converting enzyme 2 (ACE2) (Zhou,P.,et al.,Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. BioRxiv, 2020.). After the S protein of SARS-CoV-2 binds to the ACE2 receptor, it is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane (Hoffmann, M. , et al., SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell, 2020.).

The RBD region of the S protein contains major neutralizing antibody epitopes, which can stimulate B cells to produce high-titer neutralizing antibodies against RBD. In addition, the S protein also contains abundant T cell epitopes, which can induce specific CTL responses in T cells and clear virus-infected cells. Therefore, the S protein is the most critical antigen for the design of the new crown vaccine. The vast majority of vaccines currently designed have selected S protein or RBD domain protein as the core immunogen.

SARS-CoV-2 is an RNA single-stranded virus, which is prone to deletion mutations, and such mutations mostly occur in the repeat deletion regions (Recurrent deletion regions, RDRs) of the S protein. Deletion or mutation may change the conformation of the S protein, so that the antibodies induced by the previous vaccine immunization reduce the binding and neutralization of the mutant S protein, resulting in the decline of the immune effect of the vaccine and the immune escape of the virus. The early D614G mutation (B.1) enhanced the affinity of the S protein to the ACE2 receptor and quickly became a popular strain, but the mutation did not reduce the sensitivity to neutralizing antibodies (Korber, B., et al., Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2.bioRxiv,2020:p.2020.04.29.069054.; Korber,B.,et al.,Tracking Changes in SARS-CoV-2 Spike:Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell, 2020.182(4): p.812-827.e19.).

However, with the pandemic of SARS-CoV-2, five high-concern variants (Variants of Concern, VOC) have emerged in the world: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P. 1), Detla (B.1.617.2) and Omicron (B.1.1.529) and 2 kinds of variant strains (Variants of Interest, VOI): Lambda (C.37) and Mu (B.1.621). Studies have shown that these high-risk strains can increase transmissibility, exacerbate disease progression (increased hospitalization or mortality), severely reduce antibody neutralization from prior infection or immunization, reduce efficacy of treatment or vaccines, or render diagnostic testing ineffective (https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html.). Alpha spreads rapidly and can increase the risk of death by 61% (Davies, N.G., et al., Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7. Nature, 2021.). The results of the neutralizing effect study showed that the neutralizing ability of the plasma of convalescents or the serum of vaccine immunized persons remained unchanged to Alpha, but the neutralizing ability of Beta decreased significantly (Cele, S., et al., Escape of SARS- CoV-2 501Y.V2 from neutralization by convalescent plasma.2021.;Zhou,D.,et al.,Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.Cell,2021.; Tada, T., 2021. https://doi.org/10.1101/2021.02.05.430003; Wang, P., et al., Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization.bioRxiv, 2021.; Wadman, M. and J. Cohen, 2021. https://doi.org/10.1136/bmj.n296; Wu, K., et al., mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants. 2021.). Clinical results also show that Alpha has little effect on the protective effect of the vaccine, while Beta will greatly reduce the protective effect on mild disease (Madhi, S.A., et al., Efficacy of the ChAdOx1nCoV-19 Covid-19 Vaccine against the B. 1.351 Variant.2021.; Shinde,V.,et al.,Efficacy of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 Variant.New England Journal of Medicine,2021.;Abu-Raddad,L.J.,H.Chemaitelly, and A.A. Butt, Effectiveness of the BNT162b2 Covid-19 Vaccine against the B.1.1.7 and B.1.351 Variants.2021.; Karim, S.S.A., Vaccines and SARS-CoV-2 variants: the urgent need for a correlate of protection. Lancet, 2021.397(10281): p.1263-1264.). Compared with the original virus and early mutant strains, the Detla mutation has stronger transmission power, shorter incubation period, faster disease progression, and can reduce the protective effect of the vaccine. Omicron is the most severely mutated strain so far, and its S protein contains about 30 amino acid mutations. These mutations lead to large conformational changes in the S protein with large effects on infectivity and immune escape. Various studies have shown that Omicron can significantly reduce the neutralization effect induced by existing vaccines (Garcia-Beltran, W.F., et al., mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant.medRxiv, 2021: p.2021.12.14.21267755.; Dejnirattisai, W., et al., Reduced neutralization of SARS-CoV-2 omicron B.1.1.529 variant by post-immunisation serum. Lancet, 2022.399(10321): p.234- 236. Araf, Y. and F. Akter, Omicron variant of SARS-CoV-2: Genomics, transmissibility, and responses to current COVID-19 vaccines. 2022.). At present, Omicron has spread to at least 49 countries around the world, and has replaced Delta as the main epidemic strain in the world. The current vaccines are all designed based on the sequence of the early epidemic strain (its genome sequence: GenBank Accession No.NC_045512). In view of the high transmissibility of the mutant strain and the adverse impact on the protective effect of the existing vaccines, there is an urgent need for high-risk mutant strains A new type of vaccine with broad spectrum and high protective effect.

Contents of the invention

Based on the above requirements for vaccines with high protective effect on novel coronavirus SARS-CoV-2 mutant strains, the present invention provides a method for improving the ECD antigen immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strains , the method is by constructing an ECD antigen comprising at least any amino acid sequence shown in SEQ ID No:8, SEQ ID No:12 or SEQ ID No:16, or an immunogenic fragment and/or an immunogenic variant thereof, Thus ECD is a trimeric form in a stable prefusion conformation.

In one embodiment, the mutant strain contains T19I, L24S, Δ25/27, H49Y, A67V, Δ69/70, T95I, G142D, Δ143/145, Δ145-146, N211I, Δ212/ 212、V213G、G339D、R346K、R346S、S371L、S373P、S375F、T376A、D405N、R408S、K417N、N440K、L452Q、L452R、S477N、T478K、E484A、E484K、E484Q、F490S、Q493R、G496S、Q498R、N501Y、 High-risk mutant strains of at least any one of Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.

In one embodiment, the strains include B.1 strain, B.1.351 strain, B.1.1.7 strain, P.1 strain, B.1.427 strain, B.1.429 strain, B. .1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C.36.3 strain, 20I/484Q strain, BA.1 strain, BA.1.1 strain and BA.2 at least one of the strains.

In one embodiment, the ECD antigen is co-administered to the subject with one or more adjuvants selected from:

Aluminum adjuvant, oil-emulsion adjuvant, Toll-like receptor (TLR) agonist, combination of immune enhancer, microbial adjuvant, propolis adjuvant, levamisole adjuvant, liposome adjuvant, traditional Chinese medicine adjuvant and small Peptide adjuvants;

Preferably, the oil-emulsion adjuvant contains squalene;

Toll-like receptor (TLR) agonists comprising CpG or monophosphoryl lipid A (MPL) adsorbed on aluminum salts; and combinations of immunopotentiators comprising QS-21 and/or MPL.

The present invention also provides a method for improving the immunogenicity of the SARS-CoV-2 mutant strain ECD antigen immunogenicity/antigen trimer stability, the method comprises SEQ ID No: 8, SEQ ID No: 12 or SEQ ID by constructing a code A polynucleotide of at least any amino acid sequence shown in No: 16, or an immunogenic fragment and/or an immunogenic variant thereof, so as to express a trimer form of ECD in a stable prefusion conformation.

In one embodiment, the mutant strain contains T19I, L24S, Δ25/27, H49Y, A67V, Δ69/70, T95I, G142D, Δ143/145, Δ145-146, N211I, Δ212 /212, V213G, G339D, R346K, R346S, S371L, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, L452R, S477N, T478K, E484A, E484K, E484Q, N991R, G409, Q5 , Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F at least any high-risk mutant strain.

In one embodiment, the strains include B.1 strain, B.1.351 strain, B.1.1.7 strain, P.1 strain, B.1.427 strain, B.1.429 strain, B. .1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C.36.3 strain, 20I/484Q strain, BA.1 strain, BA.1.1 strain and BA.2 at least one of the strains.

The present invention provides a SARS-CoV-2 mutant strain ECD immunogenic protein/peptide with improved immunogenicity/antigen trimer stability, said immunogenic protein/peptide comprising SEQ ID No: 8, SEQ ID No:12 or SEQ ID No:16 at least one of the amino acid sequences shown in, or immunogenic fragments and / or immunogenic variants, the ECD immunogenic protein / peptide is a stable prefusion conformation of three aggregate form.

In one embodiment, the mutant strain contains T19I, L24S, Δ25/27, H49Y, A67V, Δ69/70, T95I, G142D, Δ143/145, Δ145-146, N211I, Δ212/ 212、V213G、G339D、R346K、R346S、S371L、S373P、S375F、T376A、D405N、R408S、K417N、N440K、L452Q、L452R、S477N、T478K、E484A、E484K、E484Q、F490S、Q493R、G496S、Q498R、N501Y、 High-risk mutant strains of at least any one of Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.

In one embodiment, the strains include B.1 strain, B.1.351 strain, B.1.1.7 strain, P.1 strain, B.1.427 strain, B.1.429 strain, B. .1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C.36.3 strain, 20I/484Q strain, BA.1 strain, BA.1.1 strain and BA.2 at least one of the strains.

The present invention also provides a polynucleotide encoding the ECD immunogenic protein/peptide of the SARS-CoV-2 mutant strain with improved immunogenicity/antigen trimer stability as described above; preferably, the polynucleotide comprises A nucleotide sequence of at least one of SEQ ID No: 7, SEQ ID No: 11 or SEQ ID No: 15.

The present invention provides an immunogenic composition comprising at least one immunogenic protein/peptide as described above, or at least one immunogenic protein/peptide encoding an immunogenic/antigenic trimer as described above Polynucleotides of ECD immunogenic proteins/peptides of mutant strains of SARS-CoV-2 with increased stability, and

Any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents;

Optionally, the immunogenic composition also includes an adjuvant.

Further, the present invention provides an immunogenic composition comprising the amino acid sequences shown in SEQ ID No: 12 and SEQ ID No: 16, or immunogenic fragments and/or immunogens thereof Sexual variants, or the amino acid sequences shown in SEQ ID No: 8 and SEQ ID No: 16, or immunogenic fragments and/or immunogenic variants thereof.

Further, the present invention provides an immunogenic composition, the adjuvant of the immunogenic composition is selected from one or more of the following: aluminum adjuvant, oil emulsion adjuvant, Toll-like receptor (TLR ) agonists, combinations of immunopotentiators, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposome adjuvants, traditional Chinese medicine adjuvants and small peptide adjuvants; preferably, the oil emulsion adjuvant contains squal Alkenes;

Toll-like receptor (TLR) agonists comprising CpG or monophosphoryl lipid A (MPL) adsorbed on aluminum salts; and combinations of immunopotentiators comprising QS-21 and/or MPL.

The present invention also provides the immunogenic protein/peptide as described above, polynucleotides encoding the immunogenic protein/peptide as described above and/or comprising the immunogenic protein/peptide as described above or encoding the immunogenic protein/peptide as described above The immunogenic composition of the polynucleotide of the original protein/peptide is applied to the purposes of preventing or treating the diseases caused by the mutant strain of SARS-CoV-2; meanwhile, the present invention also provides the above-mentioned immunogenic protein /peptides, polynucleotides encoding immunogenic proteins/peptides as described above and/or immunogenic combinations comprising immunogenic proteins/peptides as described above or polynucleotides encoding said immunogenic proteins/peptides The medicine is applied to the purposes of preparing vaccines or medicines for preventing or treating diseases caused by SARS-CoV-2 mutant strains.

Description of drawings

Figure 1 is a schematic diagram of the primary structure (A) and high-order structure (B, refer to PDB:6XLR) of the modified S-ECD.

Figure 2 shows the analysis results of the purity of the recombinant spike protein extracellular domain (S-ECD) trimer protein, wherein (A) is the non-reducing SDS-PAGE profile, and (B) is the SEC-HPLC profile.

Fig. 3 shows the negative staining electron microscope results of the recombinant spike protein extracellular domain (S-ECD) trimer protein.

Figure 4 shows SCTV01C-TM8 vaccine immunization after 6-8 weeks of Balb/c mice (A), 6-8 weeks of C57BL/6 mice (B) and 7-8 months old Balb/c mice (C) Test results of serum antibody titers.

Figure 5 shows that SCTV01C-TM8 vaccine immunizes 6-8 weeks of Balb/c mice (A), 6-8 weeks of C57BL/6 mice (B) and 7-8 months old Balb/c mice (C) Detection results of serum pseudovirus neutralization titer.

Figure 6 shows the statistical results of ELISpot detection of the number of T lymphocytes secreting IFN-γ (A) and IL-4 (B) in splenocytes under different peptide library stimulation conditions after SCTV01C-TM8 vaccine immunization of three mouse models.

Figure 7 shows the results of detection of SCTV01C-TM23 vaccine cynomolgus monkey immune serum antibody titer (A) and pseudovirus neutralization titer (B).

Figure 8 shows the statistical results of ELISpot detection of the number of T lymphocytes secreting IFN-γ (A) and IL-4 (B) in PBMCs under different peptide library stimulation conditions after SCTV01C-TM23 vaccine immunization in cynomolgus monkeys.

FIG. 9 shows the results of titer detection of partial antibody against Foldon in cynomolgus monkey immune serum of SCTV01C-TM23 vaccine.

Fig. 10 shows the statistical results of ELISpot detection of the number of T lymphocytes secreting IFN-γ (A) and IL-4 (B) in PBMCs under stimulation conditions of Foldon protein or 6P+Furin mutation modified peptide library.

Figure 11 shows the results of detection of SCTV01C-TM22 vaccine mouse immune serum antibody titer (A) and pseudovirus neutralization titer (B).

Fig. 12 shows the results of detection of pseudovirus broad-spectrum neutralization titer in immune sera of SCTV01C-TM22 vaccine mice.

Figure 13 shows the results of the detection of pseudovirus neutralization titer in immune sera of mice with TM8+TM23 bivalent vaccine.

Figure 14 shows the statistical results of ELISpot detection of the number of T lymphocytes secreting IFN-γ (A) and IL-4 (B) in splenocytes under different peptide library stimulation conditions after immunizing mice with TM8+TM23 bivalent vaccine.

Figure 15 shows the detection results of the neutralization titer of TM22+TM23 bivalent vaccine mouse immune sera to B.1 strain, B.1.351 strain and B.1.1.7 strain pseudovirus.

Figure 16 shows that TM22+TM23 bivalent vaccine mouse immune serum is to B.1.526 virus strain, C.37 virus strain, B.1.621 virus strain, B.1.618 virus strain, C.36.3 virus strain and 20I/484Q virus Strain pseudovirus neutralization titer test results.

Fig. 17 shows the detection results of the neutralization titer of TM22+TM23 bivalent vaccine mouse immune serum to BA.1 strain, BA.1.1 strain and BA.2 strain pseudovirus.

Detailed ways

definition

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of the present invention, the following terms are further defined.

As used herein and in the appended claims, the singular forms "a," "an," "another," and "the" include plural referents unless the context clearly dictates otherwise.

The term "comprising", "comprising" refers to the inclusion of specific components without excluding any other components. Terms such as "consisting essentially of" allow for the inclusion of other ingredients or steps that do not impair the novel or essential characteristics of the invention, ie they exclude other unrecited ingredients or steps that impair the novel or essential characteristics of the invention. The term "consisting of" means the inclusion of a specific ingredient or group of ingredients and the exclusion of all other ingredients.

The term "antigen" refers to a foreign substance that is recognized (specifically bound) by antibodies or T cell receptors, but which does not definitively induce an immune response. Exogenous substances that induce specific immunity are called "immunizing antigens" or "immunogens". By "hapten" is meant an antigen that by itself does not elicit an immune response (although a combination of several molecules of the hapten, or a combination of a hapten and a macromolecular carrier may elicit an immune response).

A "humoral immune response" is an antibody-mediated immune response and involves the introduction and production of antibodies that recognize and bind with affinity to the antigen in the immunogenic composition of the invention, a "cell-mediated immune response" is composed of T cells and and/or other white blood cell-mediated immune responses. A "cell-mediated immune response" is induced by presenting an antigenic epitope associated with a major histocompatibility complex (MHC) class I or class II molecule, CD1 or other atypical MHC-like molecule.

The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, such as a microorganism or a component thereof, which is useful for eliciting an immune response in an individual.

As used herein, "immunogenicity" means that an antigen (or an epitope of an antigen) such as the coronavirus spike protein receptor binding region or an immunogenic composition induces humoral or cell-mediated immune response, or both.

A "protective" immune response refers to the ability of an immunogenic composition to elicit a humoral or cell-mediated immune response, or both, to protect an individual from infection. The protection conferred need not be absolute, i.e., the infection need not be completely prevented or eradicated, so long as there is a statistically significant improvement relative to a control population of individuals (e.g., infected animals not administered the vaccine or immunogenic composition) . Protection may be limited to moderation of severity or rapidity of onset of symptoms of infection.

Both "immunogenic amount" and "immunologically effective amount" are used interchangeably herein to refer to an antigen or immunogenic composition sufficient to elicit an immune response (cellular (T cell) or humoral (B cell or antibody) response or both. or, as measured by standard assays known to those skilled in the art).

The effectiveness of an antigen as an immunogen can be measured, for example, by a proliferation assay, by a cell lysis assay, or by measuring the level of B cell activity.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of contiguous amino acid residues.

The terms "nucleic acid", "nucleotide" and "polynucleotide" are used interchangeably to refer to RNA, DNA, cDNA or cRNA and derivatives thereof, such as those containing modified backbones. It is to be understood that the invention provides polynucleotides comprising sequences that are complementary to the sequences described herein. A "polynucleotide" contemplated in the present invention includes the forward strand (5' to 3') and the reverse complementary strand (3' to 5'). The polynucleotides according to the invention can be prepared in different ways (e.g. by chemical synthesis, by gene cloning, etc.) and can take various forms (e.g. linear or branched, single or double stranded, or hybrids thereof , primers, probes, etc.).

The term "immunogenic protein/peptide" includes a polypeptide that is immunologically active in the sense that it is capable of eliciting a humoral and/or cell-type immune response against the protein once administered to a host. Thus, a protein fragment according to the invention comprises or consists essentially of or consists of at least one epitope or antigenic determinant. As used herein, an "immunogenic" protein or polypeptide includes the full-length sequence of the protein, an analog thereof, or an immunogenic fragment thereof. "Immunogenic fragment" refers to a fragment of a protein that contains one or more epitopes that elicit an immune response as described above.

The term "immunogenic protein/peptide" also covers deletions, additions and substitutions to sequences so long as the polypeptide functions to generate an immune response as defined herein, ie "immunogenic variants".

The SCTV01C recombinant protein vaccine provided by the present invention is transformed based on the extracellular domain (ECD, including S1 and S2 parts) of the SARS-CoV-2 spike protein. The known natural spike protein of SARS-CoV-2 has a trimeric structure. During its production and infection function, the completion of the membrane fusion process is easily detected by the RRAR site between S1 and S2. Proteases in the Golgi apparatus and on the cell surface cut open, and then S1 falls off, and the S2 structure changes from a prefusion conformation to a postfusion conformation, thereby completing membrane fusion (Cai, Y., J. Zhang, and T. Xiao, Distinct conformational states of SARS-CoV-2 spike protein. 2020.369(6511): p.1586-1592.).

In order to obtain a stable prefusion conformation ECD trimer, the present invention carried out the following three-part modification on the basis of the S protein of different strain variants:

1) It has been found that antibodies with higher neutralizing activity all bind to the S1 region (specifically, bind to the NTD and RBD regions in S1). Keeping the integrity of the S1 part is crucial for the production of neutralizing antibodies induced by the new crown vaccine. In the present invention, the Furin site is modified and removed in the SCTV01C recombinant protein vaccine, that is, the amino acid sequence at positions 679 to 688 is fixed as NSPGSASSVA, so as to reduce the possibility of S1 breaking and falling off.

2) Due to the allosteric tendency of S2 itself, the prefusion conformation of the spike protein is unstable, and the effective induction of neutralizing antibodies requires a stable prefusion conformation, which has been confirmed in RSV and HIV-1 vaccine studies (McLellan, J.S. , et al., Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science, 2013.342(6158): p.592-8.; Frey, G., et al., Distinct conformational states of HIV-1 gp41 are recognized by neutralizing and non-neutralizing antibodies. Nat Struct Mol Biol, 2010.17(12): p.1486-91.). Among the currently marketed vaccines, the S-2P (i.e. mutating amino acids at positions 986 and 987 to proline) is commonly used (Tian, J.H., et al., SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice.2021.12(1):p.372.; Mercado,N.B.,et al.,Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques.2020.586(7830):p.583-588 .; Corbett, K.S., et al., SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness. bioRxiv, 2020.). In addition, the present invention also introduces a HexaPro mutation that can effectively improve the stability without affecting its three-dimensional structure (that is, in addition to the S-2P mutation, the amino acids at positions 817, 892, 899 and 942 are mutated into proline) (Hsieh, C.L., et al., Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes. bioRxiv, 2020.). These mutation sites are all located at the N-terminus or Loop region of the α-helix in S2. After mutation to the proline (P) type with this secondary structure tendency, it can effectively reduce the allosteric tendency of S2 and stabilize the prefusion of S2 Conformation.

3) Finally, in order to further stabilize the S-ECD trimer structure, the present invention added a trimerization module T4foldon to the C-terminus of the vaccine molecule. This module is derived from the C-terminal domain of fibrin of T4 phage and has 27 amino acids. T4foldon has been used in RSV candidate vaccines, and has been shown to be safe in Phase I clinical studies (Crank, M.C., A proof of concept for structure-based vaccine design targeting RSV in humans. 2019.365(6452): p. 505-509.).

Table 1. Molecular structure design and modification of SCTV01C vaccine

Table 2. S protein mutations of SARS-CoV-2 variant strains related to the present invention

*https://www.who.int/en/activities/tracking-SARS-CoV-2-variants

**https://www.coronaheadsup.com/coronavirus/france-46-cases-of-b-1-1-7-with-e484q-mutation-in-bordeaux-coronavirus-outbreak-of-voc-20i- 484q/

***https://en.wikipedia.org/wiki/SARS-CoV-2_Omicron_variant#Mutations

****https://www.biorxiv.org/content/10.1101/2021.05.14.444076v1

Construct B.1 strain, B.1.351 and B.1.1.7 strain respectively through the expression vector of the S-ECD trimer protein antigen of above-mentioned transformation, and routinely carry out the expression recombinant S-ECD trimer protein Purity and stability analysis, preparation of corresponding vaccines, namely B.1 strain SCTV01C-TM8 vaccine, B.1.351 strain SCTV01C-TM23 vaccine and B.1.1.7 strain SCTV01C-TM22 vaccine.

The ECD trimer immunogenic protein/peptide of the present invention shows excellent immunogenicity in mice and cynomolgus monkeys, and can maintain long-term humoral and cellular immune responses.

After immunizing mice with the prepared B.1 strain SCTV01C-TM8 vaccine, the immunological assay, the immunological assay of the B.1.351 strain SCTV01C-TM23 vaccine in cynomolgus monkeys, and the B.1.1.7 strain SCTV01C- The immunological determination of TM22 vaccine in mice all shows that these three vaccines prepared by the present invention can produce sufficient titer antibody immune response in experimental animals; SCTV01C-TM8+SCTV01C-TM23 bivalent vaccine and SCTV01C-TM22+SCTV01C -The mouse immunological evaluation of the TM23 bivalent vaccine also suggests that the bivalent vaccine of the present invention has higher and similar neutralizing titers to different strains, so it has better broad-spectrum neutralization ability than the monovalent vaccine, The neutralizing titer of the bivalent vaccine against different mutant strains is much higher than that of the recovered serum against the SARS-CoV-2 strain whose genome sequence number is GenBank Accession No. MN908947.3.

Example

Example 1: Design of Trimeric Protein Antigen, Construction of Expression Vector and Protein Production of New Coronavirus Recombinant Spike Protein Extracellular Domain (S-ECD)

1.1 Table of S-ECD trimer protein (SCTV01C-TM8) based on B.1 strain sequence (EPI_ISL_406862)

Construction of the delivery vehicle

SCTV01C-TM8 contains a 3708bp gene fragment, and the target gene fragment was obtained from the template pSE-CoV2-S-ECDTM2-T4F-trimer by PCR splicing. The expression vector pGS3-2-CoV2-S-ECDTM22-T4F-trimer was constructed by the In-fusion method into the pGS3-2-SCT-1 expression vector digested with Kpn I+Not I enzymes. Using pGS3-2-CoV2-S-ECDTM8-T4F-trimer as a template, the SCTV01C-TM8 gene fragment was obtained by PCR amplification, and the expression vector of pD2535nt-HDP stable strain digested with Xba I+Asc I was constructed by In-fusion method , the expression vector pD2535nt-CoV2-S-ECDTM8-T4F-trimer of SCTV01C-TM8 was obtained.

Amplification primer

1.2 S-ECD trimer protein (SCTV01C-TM22) table based on B.1.1.7 strain sequence EPI_ISL_764238

Construction of the delivery vehicle

SCTV01C-TM22 contains a 3699bp gene fragment, and the target gene fragment was obtained from the template pD2535nt-CoV2-S-ECDTM8-T4F-trimer by PCR splicing. The expression vector pGS5-CoV2-S-ECDTM22-T4F-trimer was constructed by the In-fusion method into the pGS5-SCT-1 expression vector digested with Kpn I+Not I enzymes. Using pGS5-CoV2-S-ECDTM22-T4F-trimer as a template, the SCTV01C-TM22 gene fragment was obtained by PCR amplification, and constructed into the pD2535nt-HDP stable strain expression vector digested with Xba I+Asc I by the In-fusion method, The expression vector pD2535nt-CoV2-S-ECDTM22-T4F-trimer of SCTV01C-TM22 was obtained.

Amplification primer

1.3 S-ECD trimer protein based on B.1.351 strain sequence EPI_ISL_736940 (SCTV01C-TM23)

Construction of expression vector

SCTV01C-TM23 contains a 3699bp gene fragment. The target gene fragment was obtained from the template pD2535nt-CoV2-S-ECDTM8-T4F-trimer by PCR splicing, and constructed into pD2535nt-HDP digested with Xha I+Asc I by the In-fusion method Among the stable strain expression vectors, the expression vector pD2535nt-CoV2-S-ECDTM23-T4F-trimer of SCTV01C-TM23 was obtained.

Amplification primer

1.4 Expression and purification of S-ECD trimeric protein

The target gene constructed above was chemically transferred into HD-BIOP3(GS-) cells (Horizon), cultured in a self-developed serum-free medium, and a cell line with stable expression was obtained through MSX pressurized screening, and cultured for 14 hours. Days later, the culture supernatant was obtained by centrifugation and filtration. The culture supernatant was first captured by cation exchange chromatography (POROS XS, Thermo) and eluted with high-salt buffer; then anion chromatography (NanoGel-50Q, NanoMicro) combined mode and mixed anion chromatography (DiamondMIX-A , Borglon) flow-through mode for further purification to remove product and process-related impurities; secondly, use low pH incubation and virus removal filtration (Planova) to inactivate and remove viruses, and finally use ultrafiltration membrane packs (Millipore ) for ultrafiltration to citrate buffer. S-ECD trimer expression level >500mg/L.

Example 2: Analysis of the purity and stability of the trimer protein of the new coronavirus recombinant spike protein extracellular domain (S-ECD)

2.1 Purity analysis of recombinant S-ECD trimer protein

Place the above-mentioned purified recombinant S-ECD trimer protein stock solution in pH 6.5-7.0 buffer solution containing 1.7mM citric acid, 8mM sodium citrate, 300mM sodium chloride and 0.3mg/mL polysorbate 80 , the concentration is about 0.6mg/mL, and the purity of the primary structure is analyzed by sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS polyacrylamide gel electrophoresis, SDS-PAGE) and size-exclusion high performance liquid chromatography high performance liquid chromatograph, SEC-HPLC) to analyze its trimer content.

SDS-PAGE specific operation steps: (1) SDS-PAGE gel preparation: 3.9% stacking gel, 7.5% separating gel; (2) Boil the sample at 100°C for 2 minutes, and load 8 μg of sample after centrifugation; (3) Decolorize after Coomassie brilliant blue staining . SEC-HPLC operation step is: (1) instrument: liquid chromatography system (Agilent company, model: Agilent1260), water-soluble size exclusion chromatographic column (Sepax company, model: SRT-C SEC-500 chromatographic column); (2 ) Mobile phase: 200mM NaH ₂ PO ₄ , 100mM Arginine, pH 6.5, 0.01% isopropanol (IPA); (3) Loading amount is 80μg; (3) Detection wavelength is 280nM, analysis time is 35min, flow rate is 0.15mL /min.

The recombinant SCTV01C-TM8, SCTV01C-TM22, and SCTV01C-TM23 proteins are homotrimeric structures due to their non-covalent hydrophobic interactions. After being treated by non-reducing SDS-PAGE, monomeric molecules with a molecular weight of about 148 KDa were obtained (Fig. 2), and the purities were 98.0%, 98.8%, and 97.7%, respectively. SEC-HPLC shows that the main peaks have a purity of 96.6%, 95.5%, and 96.6%, respectively, and the proportions of aggregates and fragments are relatively small, and the average molecular weight of the main peak is 530KDa. Figure 2 is a representative test result of SCTV01C-TM8.

2.2 Morphological analysis of recombinant S-ECD trimer protein

Dynamic light scattering (DLS) and negative staining electron microscopy (Negative staining electron microscopy, EM) were used to observe the morphological characteristics of the recombinant S-ECD trimer protein.

The specific operation steps of DLS: (1) Instrument: Dynamic Light Scattering Instrument (Wyatt Technology Company, model: DynaPro NanoStar); (2) The sample volume is 50 μL; (3) After collecting the data, use Dynamics 7.1.8 software to analyze the data

The specific operation steps of negative staining electron microscope: (1) Put the carbon-coated support film copper mesh on the sealing film, drop a drop of recombinant S-ECD trimer protein sample (about 30 μm) on the support film, stay for 2-5min, and use a tape The sharp filter paper absorbs the excess solution from the edge, and the support membrane stays on the filter paper for about 10 minutes. The excess dye solution was sucked off with sharp filter paper, clamped to the filter paper to dry for 3 hours, and observed under a microscope.

The results of dynamic light scattering showed that the average molecular diameters of recombinant SCTV01C-TM8, SCTV01C-TM22, and SCTV01C-TM23 trimer proteins were 18.4nm, 19.6nm, and 18.4nm, respectively.

The results of negative staining electron microscopy confirmed that the recombinant SCTV01C-TM8, SCTV01C-TM22, and SCTV01C-TM23 trimer proteins mainly existed in the form of relatively stable pre-fusion trimers, which were different from the wild-type full-length trimer protein pre-fusion. The fusion form is similar (Figure 3), and its diameter is about 20nm, which is consistent with the diameter detected by dynamic light scattering.

2.3 Stability evaluation of recombinant S-ECD trimer protein

Thermally Accelerated Stability Evaluation

After the recombinant S-ECD trimer protein was stored at 37°C for 2 weeks (37T2W), the trimer content was analyzed by SEC-HPLC, and the data are shown in Table 2-2.

The results are shown in Table 2. After the recombinant S-ECD trimer protein was accelerated at 37°C for 2 weeks, the SEC-HPLC trimer content changes were within ±1.5%, and there was no significant increase in aggregates and fragments, showing good Thermally Accelerated Stability.

Table 2 Changes in thermally accelerated purity of recombinant S-ECD trimer protein

Freeze-thaw stability analysis

Recombinant S-ECD trimer protein was stored at -80°C for 8h, then transferred to 25°C for 0.5h (F/T-4C), and then frozen and thawed 4 times, and its trimer was analyzed by SEC-HPLC content changes.

The results are shown in Table 3. After repeated freezing and thawing of the recombinant S-ECD trimer protein, the content of the SEC-HPLC trimer was within ±1.5%, and there was no significant increase in aggregates and fragments, showing good freeze-thaw stability. stability.

Table 3 Changes in purity of recombinant S-ECD trimer protein after repeated freezing and thawing

Example 3: Immunological evaluation of B.1 strain SCTV01C-TM8 vaccine in different types of mice

3.1 Vaccine preparation

The purified SCTV01C-TM8 protein was pre-diluted with PBS to 20 μg/mL or 60 μg/mL, and the diluted antigen was mixed with MF59 (source: China Cell Engineering Co., Ltd., the same below) in equal volumes to obtain a final antigen concentration of 10 μg/mL. mL or 30 μg/mL of finished vaccine containing MF59.

3.2 Immunization of mice

6-8 weeks of Balb/c mice, 6-8 weeks of C57BL/6 mice, 7-8 months old Balb/c mice (source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), intramuscular injection of 0.1mL containing The finished vaccine with MF59 adjuvant. Three types of mice were immunized with 1 μg, 3 μg and 3 μg of antigens respectively. A total of 3 immunizations were carried out, and the immunization interval was 3 weeks. Two weeks after the first immunization, orbital blood was collected every other week, and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immune analysis.

3.3 Determination of antibody titer and neutralizing titer of mouse immune serum

Coat 100 μL/well of SCTV01C-TM8 protein at a concentration of 5 μg/mL on a 96-well plate, and coat overnight at 2-8°C. After the enzyme plate was washed and patted dry, 320 μL/well of blocking solution containing 2% BSA was added, and blocked at room temperature for more than 1 h. Use the TBST sample diluent containing 0.1% BSA to serially dilute the SCTV01C-TM8 mouse immune serum (such as 8000×, 16000×, 32000×, 64000×, 128000×, 256000×, 512000×, etc.), and dilute in the same gradient Serum from unimmunized mice was used as negative control serum. Add 100 μL/well of serially diluted serum to the 96-well ELISA plate, and incubate at room temperature for 1-2 hours. The plate was washed 3 times, 100 μL/well of 80 ng/mL rabbit anti-mouse IgG F(ab) ₂ /HRP detection secondary antibody (source: Jackson ImmunoResearch, the same below) was added, and incubated at room temperature for 1 h. Wash the plate 5 times, add the substrate chromogenic solution to develop the color for 10-15 minutes, and read the OD ₄₅₀ with a microplate reader after the termination of 2M H ₂ SO ₄ to calculate the immune antibody titer. Antibody titer = greater than the maximum dilution factor of negative serum OD ₄₅₀ ×2.1.

Add 50 μL/holes of immune serum of different dilutions to 96-well plates, then add 50 μL/wells of 100TCID ₅₀ pseudoviruses of SARS-CoV-2 strains with GenBank Accession No.MN908947.3 (pseudoviruses are based on The replication-defective vesicular stomatitis virus (VSV△G-Luc-G) in which the VSV-G protein gene in the viral genome was replaced by a luciferase reporter gene was used as a vector, and carried out in a cell line expressing Spike and its mutant proteins Amplification preparation, prepared by Shenzhou Cell Engineering Co., Ltd., the same below), mixed evenly and placed in a 37°C, 5% CO ₂ incubator for 1h incubation. Serum-free cell wells containing pseudovirus were used as positive controls, and cell wells without serum and pseudoviruses were used as negative controls. After the incubation, 3×10 ⁴ 293FT-ACE2 cells were inoculated at 100 μL/well, mixed well, and placed in a 37° C., 5% CO ₂ incubator for static culture for about 20 hours. After the culture, remove the culture supernatant, add 50 μL/well of 1×Passive lysis buffer, and mix well to lyse the cells. 40 μL/well was transferred to a 96-well all-white chemiluminescent plate, and 40 μL/well of luciferase substrate was added using a LB960 microplate luminescence detector to detect the luminescence value (RLU) and calculate the neutralization rate. Neutralization rate%=(positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs)×100%, IC ₅₀ was calculated according to Reed-Muench formula, which was NAT ₅₀ .

The antibody titer results after immunization are shown in Figure 4. SCTV01C-TM8 was tested in 6-8 week Balb/c mice, 6-8 week C57BL/6 mice and 7-8 month old Balb/c mice. All models can induce high-titer antibody immune responses. The pseudovirus neutralization titer results of the SARS-CoV-2 strain whose genome sequence number is GenBank Accession No. MN908947.3 are shown in Figure 5. SCTV01C-TM8 can induce high-titer pseudoviruses in all three mouse models Neutralizing antibodies. After immunization, the neutralizing titer of the pseudovirus is significantly higher than that of the SARS-CoV-2 strain whose genome sequence number is GenBank Accession No. MN908947.3 by the serum (HCS) of recovered patients with new crown infection, 3 free for 7 days The neutralizing titers of HCS were 33.3 times, 13.9 times and 3.9 times respectively.

3.4 Detection of vaccine-induced cellular immune response

Mouse splenocytes were isolated, and 100 μL/well of mouse splenocytes were inoculated on pre-treated ELISpot well plates (source: Mabtech, the same below), at a cell inoculation density of 2×10 ⁵ cells/well. Then 100 μL/well was added to RBD, S1, S2 or S protein peptide library with a final concentration of 2 μg/mL (15 amino acids/peptide, overlapping 11 amino acids, source: Beijing Zhongke Yaguang Biotechnology Co., Ltd. Synthesis, the same below ), and incubated in a 37°C, 5% CO ₂ incubator for about 20h. After the incubation, the cell supernatant of the ELISpot well plate was removed, the plate was washed 5 times with PBS, and then 100 μL/well of the diluted detection antibody was added. After incubation for 2 hours, the plate was washed 5 times with PBS, and diluted Streptavidin-ALP (1:1000) was added to 100 μL/well. After incubation at room temperature for 1 h, the plate was washed 5 times with PBS, and then 100 μL/well of BCIP/NBT-plus substrate filtered with a 0.45 μm filter membrane was added. Keep away from light at room temperature for 10-30 minutes to develop color until clear spots appear, and stop with deionized water. Place the ELISpot well plate in a cool place at room temperature, wait for it to dry naturally, and analyze the results with an enzyme-linked spot analyzer. The number of antigen-specific IFN-γ or IL-4 secreting positive T cells was represented by SFC (Spot-forming cells) per 10 ⁶ mouse splenocytes, and the GrapPad Prism software was used for data statistics.

The results are shown in Figure 6. SCTV01C-TM8 can induce higher Th1 (IFN-γ) and Th2 (IL- 4) Cellular response.

Example 4: Immunological evaluation of B.1.351 strain SCTV01C-TM23 vaccine in cynomolgus monkeys

4.1 Vaccine preparation

The purified SCTV01C-TM23 protein was pre-diluted with PBS to 120 μg/mL, and the diluted antigen was mixed with MF59 in equal volumes to obtain a finished vaccine containing MF59 with a final antigen concentration of 60 μg/mL.

4.2 Immunization of cynomolgus monkeys

Cynomolgus monkeys (source: Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.), intramuscularly injected 0.5 mL of finished vaccine containing MF59 adjuvant, with an antigenic amount of 30 μg. A total of 2 immunizations were performed with an interval of 3 weeks. Two weeks after the first immunization, venous blood was collected every other week, and non-anticoagulant centrifuge tubes were pre-incubated in ice water before use. After the blood sample was collected, it was transferred to a centrifuge tube without anticoagulant for temporary storage, and then centrifuged at 3000×g for 10 min at 2-8°C. Separated serum samples were subjected to subsequent serological immunoassays. Blood samples collected using anticoagulant centrifuge tubes were routinely isolated from peripheral blood lymphocytes (PBMCs) for cellular immunoassays.

4.3 Determination of antibody titer and neutralizing titer of cynomolgus monkey immune serum

Anti-SCTV01C-TM23 or Foldon part-specific IgG antibodies in serum of cynomolgus monkeys were detected by ELISA. Dilute SCTV01C-TM23 with coating solution to 2 μg/mL, add 100 μL/well into the microplate, and incubate overnight at 2-8°C. Wash the plate 3 times and pat dry, add 300 μL/well of 2% casein-PBST blocking solution, and block at room temperature for at least 1 h. Wash the plate 3 times and pat dry, dilute the sample to 1000 times with 0.1% casein-PBST as the sample diluent, and then use 1‰ mixed blank cynomolgus serum to serially dilute the immune serum (such as 8000×, 16000×, 32000×, 64000×, 128000×, 256000×, 512000×, etc.), add 100 μL/well according to the layout, seal the plate, and incubate at room temperature for about 2 hours with shaking. Wash the plate 3 times and pat dry, dilute the secondary antibody Goat pAb to Mk IgG (HRP) (source: Abcam, the same below) to 15ng/mL with 0.5% casein-PBST secondary antibody diluent, add 100μL/well to the microtiter plate Inside, incubate at room temperature in the dark for about 1 hour; wash the plate 3 times, mix the chromogenic solutions A and B at a ratio of 1:1, apply 100 μL/well, and incubate at room temperature in the dark for about 15 minutes; add 50 μL/well of stop solution, The 450nm wavelength of the microplate reader is read, and the antibody titer is calculated. Antibody titer = greater than the maximum dilution factor of negative serum OD ₄₅₀ ×2.1.

Add 50 μL/well of immune sera with different dilutions to the 96-well plate. Then add 100-200 TCID ₅₀ of B.1.351 strain pseudovirus at 50 μL/well, mix well, and then place in a 37° C., 5% CO2 incubator to incubate for 1 hour. Serum-free cell wells containing pseudovirus were used as positive controls, and cell wells without serum and pseudoviruses were used as negative controls. After the incubation, 100 μL/well was inoculated with 2×10 ⁴ Huh-7 cells, mixed evenly, and placed in a 37° C., 5% CO ₂ incubator for static culture for about 20 h. After the culture, remove the culture supernatant, add 50 μL/well of 1×Passive lysis buffer, and mix well to lyse the cells. 40 μL/well was transferred to a 96-well all-white chemiluminescent plate, and 40 μL/well of luciferase substrate was added using a LB960 microplate luminescence detector to detect the luminescence value (RLU) and calculate the neutralization rate. Neutralization rate%=(positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs)×100%, IC ₅₀ was calculated according to the Reed-Muench formula, which was the neutralization titer NAT ₅₀ .

The results are shown in Figure 7. SCTV01C-TM23 can induce a high-titer antibody immune response in cynomolgus monkeys. Neutralization of the pseudovirus of the B.1.351 strain showed that the neutralization titer gradually increased with time after immunization, and at 2 The titer peak was reached 7 days to 14 days after immunization.

4.4 Detection of vaccine-induced cellular immune response

The literature shows that there are conserved T cell epitopes among different variants of SARS-CoV-2 (Redd, AD, et al., CD8+T cell responses in COVID-19 convalescent individuals target conserved epitopes from multiple prominent SARS-CoV -2 circulating variants.medRxiv,2021:p.2021.02.11.21251585.;Tarke,A.,et al.,Negligible impact of SARS-CoV-2 variants on CD4+and CD8+T cell reactivity in COVID-19 exposed donors and vaccinees.bioRxiv, 2021: p.2021.02.27.433180.), so the polypeptide library of the SARS-CoV-2 strain sequence whose genome sequence number is GenBank Accession No. MN908947.3 is still used for T cell immune detection. Monkey PBMCs were isolated by density gradient centrifugation, and 100 μL/well of PBMCs cells were inoculated on the pre-treated ELISpot well plate at a cell seeding density of 2.5×10 ⁵ cells/well, and then RBD with a final concentration of 2 μg/mL was added to 100 μL/well , S1 or S protein peptide library, and incubated in a 37°C, 5% CO ₂ incubator for about 20 hours. After the incubation, the cell supernatant of the ELISpot well plate was removed, the plate was washed 5 times with PBS, and then 100 μL/well of the diluted detection antibody with a final concentration of 1 μg/mL was added. After incubation for 2 hours, the plate was washed 5 times with PBS, 100 μL/well of diluted Streptavidin-ALP (1:1000) was added, and the plate was washed 5 times with PBS after incubation at room temperature for 1 hour. Then 100 μL/well of BCIP/NBT-plus substrate filtered with a 0.45 μm filter membrane was added, and the color was developed for 10-30 minutes at room temperature in the dark until clear spots appeared and terminated with deionized water. Place the ELISpot well plate in a cool place at room temperature, wait for it to dry naturally, and analyze the results with an enzyme-linked spot analyzer. The number of antigen-specific IFN-γ or TL-4 secreting positive T cells was represented by SFC (Spot-forming cells) per 10 ⁶ PBMC cells, and the GrapPad Prism software was used for data statistics.

The results are shown in Figure 8, SCTV01C-TM23 can induce Th1 (IFN-γ) and Th2 (IL-4) cell responses against RBD, S1, and S polypeptide libraries in cynomolgus monkeys after 2 immunizations and 7 days.

4.5 Monkey immunogenicity of Foldon and mutation points in S-ECD trimer molecule

The Foldon-containing RSV F recombinant protein (RSV-F-Foldon) was used as the coating antigen to detect the antibody titer against Foldon after SCTV01C-TM23 was immunized with cynomolgus monkeys with reference to Example 4.3. The results are shown in Figure 9, the immunogenicity induced by Foldon in the S-ECD trimer molecule is very weak, and the immune titer of SCTV01C-TM23 in cynomolgus monkeys at different time points is 54-76 times that of Foldon.

The RSV-F-Foldon protein or the peptide library containing the "6P" mutation and the Furin site mutation were used to detect the effect of the introduction of Foldon and the mutation modification on the immune response of T cells with reference to Example 4. The results are shown in Figure 10. After 2 immunizations and 7 days, Foldon had a very low T cell immune response in cynomolgus monkeys, and the "6P" and Furin site mutations had no effect on the cellular immune response.

In summary, the three modifications made to stabilize the trimer structure of S-ECD have weak immunogenicity and little interference with S-ECD immunity.

Example 5: Immunological evaluation of B.1.1.7 strain SCTV01C-TM22 vaccine in mice

5.1 Vaccine preparation

The purified SCTV01C-TM22 protein was pre-diluted to 20 μg/mL with PBS, and the diluted antigen was mixed with MF59 in equal volumes to obtain a finished vaccine containing MF59 with a final antigen concentration of 10 μg/mL.

5.2 Immunization of mice

C57BL/6 mice (source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) at 6-8 weeks were intramuscularly injected with 0.1 mL of the finished vaccine containing MF59 adjuvant, and the amount of antigen was 1 μg. A total of 3 immunizations were performed with an interval of 2 weeks. Two weeks after the first immunization, orbital blood was collected every other week, and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immune analysis.

5.3 Determination of antibody titer and neutralizing titer of mouse immune serum

Using SCTV01C-TM22 as the coating antigen, refer to Example 3.3 to detect the antibody titer after SCTV01C-TM22 immunized C57BL/6 mice.

Add 50 μL/well of 2-immune 7-day immune serum with different dilutions into a 96-well plate, then add 100-200 TCID ₅₀ of B.1.1.7 strain pseudovirus at 50 μL/well, mix well and place at 37°C, 5% CO ₂ incubator for 1h. Serum-free cell wells containing pseudovirus were used as positive controls, and cell wells without serum and pseudoviruses were used as negative controls. After the incubation, 100 μL/well was inoculated with 2×10 ⁴ Huh-7 cells, mixed evenly, and placed in a 37° C., 5% CO ₂ incubator for static culture for about 20 h. After the culture, remove the culture supernatant, add 50 μL/well of 1×Passive lysis buffer, and mix well to lyse the cells. 40 μL/well was transferred to a 96-well all-white chemiluminescent plate, and 40 μL/well of luciferase substrate was added using a LB960 microplate luminescence detector to detect the luminescence value (RLU) and calculate the neutralization rate. Neutralization rate%=(positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs)×100%, IC ₅₀ was calculated according to the Reed-Muench formula, which was the neutralization titer NAT ₅₀ .

B.1.1.7 Strain pseudovirus neutralizing titer results As shown in Figure 11, SCTV01C-TM22 can induce high titers of pseudovirus neutralizing antibodies in C57BL/6 mice. The neutralizing titer after immunization is higher than the neutralizing titer of the SARS-CoV-2 strain whose genome sequence number is GenBank Accession No. MN908947.3 by the serum (HCS) of recovered patients with new crown infection. The neutralizing titer is 56.5 times that of HCS.

5.4 Broad-spectrum neutralizing activity of SCTV01C-TM22 vaccine

With reference to embodiment 5.3 detection SCTV01C-TM22 vaccine 2 immune sera for 7 days to other variant strains (B.1 strain, B.1.351 strain, P.1 strain and B.1.429 strain and/or B.1.427 strain) pseudovirus neutralization titer. The results are shown in Figure 12, SCTV01C-TM22 vaccine immune serum still has higher neutralizing titer to B.1 strain, but to B.1.351 strain, P.1 strain, and B.1.429 strain and /or B.1.427 strain neutralization titer is greatly reduced.

The neutralizing activity test results of SCTV01C-TM22 vaccine showed that the monovalent vaccine could not produce high-titer neutralizing antibodies against mutant strains, and had poor broad-spectrum neutralizing activity. In order to cope with virus mutation and enhance the cross-protection ability of the vaccine against the original strain and multiple mutant strains, the following examples prepared bivalent vaccines containing S-ECD proteins of different mutant strains, and tested the broad-spectrum of the bivalent vaccine. Immunological evaluation was carried out with the ability.

Example 6: Immunological evaluation of SCTV01C-TM8+SCTV01C-TM23 bivalent vaccine in mice

6.1 Vaccine preparation

The purified SCTV01C-TM8 and SCTV01C-TM23 proteins were pre-diluted to 20 μg/mL with PBS, and the diluted antigen was mixed with MF59 in equal volumes to obtain a finished monovalent vaccine containing MF59 with a final antigen concentration of 10 μg/mL.

The SCTV01C-TM8 and SCTV01C-TM23 proteins were pre-diluted with PBS to 40 μg/mL, and the diluted antigens were mixed in equal volumes to obtain a mixed antigen sample with a final antigen concentration of 20 μg/mL. The mixed antigen sample was mixed with MF59 in equal volumes to obtain the finished bivalent vaccine TM8+TM23 containing MF59 with the final antigen concentration of SCTV01C-TM8 and SCTV01C-TM23 both being 10 μg/mL.

6.2 Immunization of mice

C57BL/6 mice at 6-8 weeks (source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), intramuscularly injected with 0.1 mL of the monovalent or bivalent vaccine product containing MF59 adjuvant, the antigen content of the monovalent vaccine is 1 μg, and the bivalent vaccine The amount of antigen was 1 μg, respectively. A total of 3 immunizations were carried out, and the immunization interval was 3 weeks. Two weeks after the first immunization, orbital blood was collected every other week, and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immune analysis.

6.3 Determination of the neutralization titer of TM8+TM23 bivalent vaccine immune serum to different mutant strains

With reference to Example 5.3, detect the immune serum of the bivalent vaccine for 2 immunizations for 7 days to different mutant strains (B.1 strain, B.1.351 strain, B.1.1.7 strain, P.1 strain, and B.1.429 virus strain and/or B.1.427 strain) pseudovirus neutralization titer. The results are shown in Figure 13, the SCTV01C-TM8 monovalent vaccine has a higher neutralizing titer to the B.1 strain and the B.1.1.7 strain pseudovirus, but to the B.1.351 strain and the P.1 strain pseudovirus The neutralizing titer is low, and the reduction factor is about 5-11 times of the neutralizing titer of the B.1 strain. SCTV01C-TM23 monovalent vaccine has higher neutralizing titer to B.1.351 strain and P.1 strain pseudovirus, but to B.1 strain, B.1.1.7 strain, and B.1.429 strain and/or Or the neutralizing titer of the B.1.427 strain pseudovirus is low, and the reduction factor is about 4 to 12 times of the neutralizing titer of the B.1.351 strain. The TM8+TM23 bivalent vaccine has high and similar neutralizing titers against different strains, so it has better broad-spectrum neutralizing ability than the monovalent vaccine. The neutralizing titer of the bivalent vaccine against different mutant strains is much higher than that of the recovered serum against the SARS-CoV-2 strain whose genome sequence number is GenBank Accession No. MN908947.3.

6.4 Detection of vaccine-induced cellular immune response

Use the RBD, S1, S peptide library of the SARS-CoV-2 strain sequence with the genome sequence number GenBank Accession No.NC_045512 and the SARS-CoV-2 strain S peptide library with the genome sequence number GenBank Accession No.MN908947.3 Refer to Example 3.4 to detect the T cell immune response of mice immunized with TM8+TM23 bivalent vaccine for 3 days and 7 days after using the mixed peptide library of B.1.351 strain differential polypeptide (TM23-mix). The results are shown in Figure 14. The peptide library of the SARS-CoV-2 strain whose genome sequence number is GenBank Accession No. MN908947.3 has a higher T cell response after stimulating the splenocytes of mice immunized with monovalent vaccine and bivalent vaccine. And T cell immune response is similar. The S-peptide library and the S+TM23-mix mixed peptide library stimulated similar T cell responses, indicating that the differential polypeptides of the B.1.351 strain had no T-cell immune response, and further proved that there were conserved T-cell epitopes among mutant strains.

Example 7: Immunological evaluation of SCTV01C-TM22+SCTV01C-TM23 bivalent vaccine in mice

7.1 Vaccine preparation

The purified SCTV01C-TM22 and SCTV01C-TM23 proteins were pre-diluted with PBS to 20 μg/mL, and the diluted antigen was mixed with MF59 in equal volumes to obtain a finished monovalent vaccine containing MF59 with a final antigen concentration of 10 μg/mL.

The SCTV01C-TM22 and SCTV01C-TM23 proteins were pre-diluted with PBS to 40 μg/mL, and the diluted antigens were mixed in equal volumes to obtain a mixed antigen sample with a final antigen concentration of 20 μg/mL. The mixed antigen sample was mixed with MF59 in equal volumes to obtain the finished bivalent vaccine TM22+TM23 containing MF59 with the final antigen concentration of SCTV01C-TM22 and SCTV01C-TM23 both being 10 μg/mL.

7.2 Immunization of mice

C57BL/6 mice at 6-8 weeks (source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), intramuscularly injected with 0.1 mL of the monovalent or bivalent vaccine product containing MF59 adjuvant, the antigen content of the monovalent vaccine is 1 μg, and the bivalent vaccine The amount of antigen was 1 μg (the amount of antigen in the bivalent vaccine in Example 7.5 was 0.5 μg). A total of 2 immunizations were carried out, and the immunization interval was 2 weeks. Two weeks after the first immunization, orbital blood was collected every other week, and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immune analysis.

7.3 Determination of the neutralization titer of SCTV01C-TM22+TM23 bivalent vaccine immune serum to different mutant strains (I)

Refer to Example 5.3 to detect the pseudovirus neutralization titer of the bivalent vaccine 2-immune 7-day immune serum to different mutant strains (B.1 strain, B.1.351 strain and B.1.1.7 strain). The results are shown in Figure 15. SCTV01C-TM22 monovalent vaccine has a higher neutralizing titer to B.1.1.7 strains and B.1 strain pseudoviruses, but the neutralizing ability to B.1.351 strains decreased by about It is 8.8 times of the neutralizing titer of B.1.1.7 strain. SCTV01C-TM23 monovalent vaccine has higher neutralizing titer to B.1.351 strain, but lower neutralizing potency to B.1.1.7 strain and B.1 strain, and the reduction times are B.1.351 strain neutralization The potency is 6.4 times and 5.1 times. The TM22+TM23 bivalent vaccine has high and similar neutralizing titers against different strains, so it has better broad-spectrum neutralizing ability than the monovalent vaccine. The neutralizing titer of the bivalent vaccine against different mutant strains is much higher than that of the recovered serum against the SARS-CoV-2 strain whose genome sequence number is GenBank Accession No. MN908947.3.

7.4 Determination of the neutralization titer of SCTV01C-TM22+TM23 bivalent vaccine immune serum to different mutant strains (II)

With reference to Example 5.3, the immune serum of the bivalent vaccine was tested for 2 immunizations for 7 days to different mutant strains of SARS-CoV-2 (B.1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C. 36.3 strain and 20I/484Q strain) pseudovirus neutralization titer. The results are shown in Figure 16, the SCTV01C-TM22+TM23 bivalent vaccine immunized mouse serum against B.1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C.36.3 strain and 20I/484Q strain pseudovirus neutralizing antibody titer NAT ₅₀ geometric mean (GMT) compared with monovalent vaccine SCTV01C-TM22 and SCTV01C-TM23 all have a certain degree of increase (increasing multiples are 2.8 times, 2.6 times, respectively. times, 3.6 times, 4.2 times, 2.1 times, 0.8 times; and 1.8 times, 3.2 times, 1.5 times, 1.7 times, N/A, 2.0 times), indicating that the SCTV01C-TM22+TM23 bivalent vaccine has Higher and similar neutralizing titer, so it has better broad-spectrum neutralizing ability than monovalent vaccine.

7.5 Determination of Neutralizing Potency of SCTV01C-TM22+TM23 Bivalent Vaccine Immune Sera to Different Mutant Strains (III)

With reference to Example 5.3, detect the pseudovirus neutralizing titer of the bivalent vaccine for 2 immune sera in 21 days to different mutant strains (BA.1 strain, BA.1.1 strain, BA.2 strain) of SARS-CoV-2Omicron. The results are shown in Figure 17. The mouse serum of SCTV01C-TM22+TM23 bivalent vaccine immunized group against Omicron BA.1 strain, BA.1.1 strain, BA.2 strain pseudovirus neutralizing antibody titer NAT ₅₀ geometric mean Compared with the monovalent vaccine TM8, the GMT value (GMT) has a certain degree of increase (increasing multiples are 18.3 times, 25.3 times, 10.4 times respectively), indicating that the SCTV01C-TM22+TM23 bivalent vaccine has different effects on SARS-CoV-2Omicron variation. All strains have higher neutralizing titers, so they have better broad-spectrum neutralizing ability than monovalent vaccines.

In summary, compared with monovalent vaccines, bivalent vaccines have broad-spectrum neutralization capabilities against different mutant strains, and are expected to produce cross-protection capabilities against multiple mutant strains and improve the protection rate against mutant strain infection.

Although the foregoing has described the present invention in detail by means of illustrations and examples, its purpose is to facilitate understanding, and those skilled in the art can obviously make various modifications and improvements to the technical solutions of the present invention without departing from the additional the spirit or scope of the claims.

All documents cited in this specification are incorporated by reference in their entirety.

sequence list

Claims (16)

1. A method for improving the immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strain ECD antigen, the method comprises SEQ ID No:8, SEQ ID No:12 or shown in SEQ ID No:16 by constructing At least any amino acid sequence, or an ECD antigen of an immunogenic fragment and/or an immunogenic variant thereof, such that the ECD is a trimeric form in a stable prefusion conformation.
2. The method of claim 1, wherein the mutant strain contains T19I, L24S, Δ25/27, H49Y, A67V, Δ69/70, T95I, G142D, Δ143/145, Δ145-146, N211I, Δ212/ 212、V213G、G339D、R346K、R346S、S371L、S373P、S375F、T376A、D405N、R408S、K417N、N440K、L452Q、L452R、S477N、T478K、E484A、E484K、E484Q、F490S、Q493R、G496S、Q498R、N501Y、 High-risk mutant strains of at least any one of Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.
3. The method of claim 1 or 2, wherein the strain comprises B.1 strain, B.1.351 strain, B.1.1.7 strain, P.1 strain, B.1.427 strain, B.1.429 strain , B.1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C.36.3 strain, 20I/484Q strain, BA.1 strain, BA.1.1 strain and BA .2 At least one of the strains.
4. The method of claim 1, wherein the ECD antigen is administered to the subject together with one or more adjuvants selected from the group consisting of aluminum adjuvant, oil-emulsion adjuvant, Toll-like receptor (TLR) agonist, immune enhancer Combination of microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposome adjuvants, traditional Chinese medicine adjuvants and small peptide adjuvants;

   Preferably, the oil-emulsion adjuvant contains squalene;

   Toll-like receptor (TLR) agonists comprising CpG or monophosphoryl lipid A (MPL) adsorbed on aluminum salts; and combinations of immunopotentiators comprising QS-21 and/or MPL.
5. A method for improving the immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strain ECD antigen, the method is by constructing a code comprising SEQ ID No: 8, SEQ ID No: 12 or SEQ ID No: 16 A polynucleotide of at least any one of the amino acid sequences shown, or an immunogenic fragment and/or an immunogenic variant thereof, thereby expressing a trimer form of ECD in a stable prefusion conformation.
6. The method of claim 5, wherein the mutant strain contains T19I, L24S, Δ25/27, H49Y, A67V, Δ69/70, T95I, G142D, Δ143/145, Δ145-146, N211I, Δ212/ 212、V213G、G339D、R346K、R346S、S371L、S373P、S375F、T376A、D405N、R408S、K417N、N440K、L452Q、L452R、S477N、T478K、E484A、E484K、E484Q、F490S、Q493R、G496S、 Q498R、N501Y、 High-risk mutant strains of at least any one of Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.
7. The method of claim 5 or 6, wherein the strain comprises B.1 strain, B.1.351 strain, B.1.1.7 strain, P.1 strain, B.1.427 strain, B.1.429 strain , B.1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C.36.3 strain, 20I/484Q strain, BA.1 strain, BA.1.1 strain and BA .2 At least one of the strains.
8. A kind of SARS-CoV-2 mutant strain ECD immunogenic protein/peptide that immunogenicity/antigen trimer stability improves, is characterized in that, this immunogenic protein/peptide comprises SEQ ID No:8, SEQ ID No:12 or SEQ ID No:16 at least one of the amino acid sequences shown in, or immunogenic fragments and / or immunogenic variants, the ECD immunogenic protein / peptide is a stable prefusion conformation of three aggregate form.
9. The immunogenic protein/peptide of claim 8, wherein the mutant strain contains T19I, L24S, Δ25/27, H49Y, A67V, Δ69/70, T95I, G142D, Δ143/145, Δ145-146, N211I, △212/212, V213G, G339D, R346K, R346S, S371L, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, L452R, S477N, T478K, E484A, G, E484S, E484S, E498 , Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F at least any high-risk mutant strain.
10. The immunogenic protein/peptide of claim 8 or 9, wherein the strain comprises B.1 strain, B.1.351 strain, B.1.1.7 strain, P.1 strain, B.1.427 strain, B.1.429 strain, B.1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C.36.3 strain, 20I/484Q strain, BA.1 strain, BA. At least one of the 1.1 strain and the BA.2 strain.
11. A polynucleotide encoding the immunogenic protein/peptide of claim 8,

    Preferably, it comprises at least any one of the nucleotide sequences shown in SEQ ID No: 7, SEQ ID No: 11 or SEQ ID No: 15.
12. An immunogenic composition, characterized in that, comprising

    at least one immunogenic protein/peptide as claimed in claim 8, or

    at least one polynucleotide as claimed in claim 11, and

    Any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents;

    Optionally, an adjuvant is included.
13. The immunogenic composition of claim 12, comprising

    the amino acid sequences shown in SEQ ID No: 12 and SEQ ID No: 16, or immunogenic fragments and/or immunogenic variants thereof, or

    The amino acid sequences shown in SEQ ID No: 8 and SEQ ID No: 16, or immunogenic fragments and/or immunogenic variants thereof.
14. The immunogenic composition according to claim 12 or 13, characterized in that the adjuvant is selected from one or more of the following:

    Aluminum adjuvant, oil-emulsion adjuvant, Toll-like receptor (TLR) agonist, combination of immune enhancer, microbial adjuvant, propolis adjuvant, levamisole adjuvant, liposome adjuvant, traditional Chinese medicine adjuvant and small Peptide adjuvants;

    Preferably, the oil-emulsion adjuvant contains squalene;

    Toll-like receptor (TLR) agonists comprising CpG or monophosphoryl lipid A (MPL) adsorbed on aluminum salts; and combinations of immunopotentiators comprising QS-21 and/or MPL.
15. The immunogenic protein/peptide of claim 8, the polynucleotide of claim 11 and the immunogenic composition of any one of claims 12-14 prevent or treat SARS-CoV-2 mutant strains The use of the disease caused.
16. The immunogenic protein/peptide according to claim 8, the polynucleotide according to claim 11 and the immunogenic composition according to any one of claims 12-14 are used in the preparation of preventing or treating SARS-CoV-2 mutations Use in vaccines or medicines for diseases caused by strains.

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