WO2023160654A1

WO2023160654A1 - Preparation and use of recombinant multicomponent sars-cov-2 trimeric protein vaccine capable of inducing broad-spectrum neutralizing activity

Info

Publication number: WO2023160654A1
Application number: PCT/CN2023/078135
Authority: WO
Inventors: 谢良志; 孙春昀; 张延静
Original assignee: 神州细胞工程有限公司
Priority date: 2022-02-25
Filing date: 2023-02-24
Publication date: 2023-08-31
Also published as: CN118076646A

Abstract

The present invention relates to the field of molecular vaccinology. Provided in the present invention is a recombinant multicomponent SARS-CoV-2 trimeric protein vaccine capable of inducing a broad-spectrum neutralizing activity. Recombinant protein ingredients include, but are not limited to, homotrimeric proteins formed by means of introducing mutation sites and trimeric auxiliary structures to the extracellular domains (ECD) of spike proteins (S proteins) of Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and BA.1 (B.1.1.529.1). The multicomponent vaccine contains an ECD trimeric protein of the above variants, either alone or in any combination, and a pharmaceutically acceptable adjuvant. The vaccine combination shows excellent immunogenicity in mice, while also maintaining long-term humoral immunity and cellular immune responses. The multicomponent SARS-CoV-2 trimeric protein vaccine can be used for preventing infection-related diseases caused by infections with SARS-CoV-2 and variants thereof.

Description

Preparation and application of a recombinant multi-component novel coronavirus trimeric protein vaccine capable of inducing broad-spectrum neutralization activity

Cross References to Related Applications

This application claims the benefit of Chinese patent application 202210184528.3 filed on February 25, 2022, the contents of which are incorporated herein by reference.

technical field

The invention relates to the field of molecular vaccinology, and relates to the preparation and application of a recombinant multi-component novel coronavirus trimeric protein vaccine capable of inducing broad-spectrum neutralization activity.

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. Since the SARS-CoV-2 pandemic, more than 200 new crown vaccines have been developed by various countries. As of December 2, 2022, 50 vaccines have been approved for use or conditional use worldwide, and another 242 vaccines have entered clinical research (https://covid19.trackvaccines.org/vaccines/).

Angiotensin-converting enzyme 2 (ACE2) is a common host cell receptor protein of SARS-CoV-2 and SARS-CoV ^[1] . The trimeric spike protein (Spike) of the virus binds to the ACE2 receptor and 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 ^{[2 ]} . The S protein is the main component of the viral envelope and plays an important role in receptor binding, fusion, virus entry and host immune defense. 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, prone to deletion mutations, and such mutations mostly occur in the recurrent deletion regions (RDRs) of the S protein. Deletion or mutation may change the conformation of the S protein, reducing the binding and neutralization of the mutant S protein by antibodies induced by previous vaccine immunization, resulting in a decline in the immune effect of the vaccine and 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 ^[3,4] . 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), Delta (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 therapeutic or vaccine efficacy, or render diagnostic testing ineffective ^{[ 5]} . Alpha spreads rapidly and can increase the risk of death by 61% ^[6] . The results of the neutralization effect study showed that the neutralization ability of the plasma of convalescents or the serum of vaccine immunized persons remained basically unchanged to Alpha, but the neutralization ability of Beta decreased significantly ^[7-12] . 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 ^[13-16] . Compared with the original virus and early mutant strains, the Delta 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, which have a great impact on infectivity and immune escape. Various studies have shown that Omicron It can greatly reduce the neutralization effect induced by existing vaccines ^[17-19] . 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 early epidemic strains (its genome sequence: GenBank Accession No.NC_045512). In view of the high transmissibility of mutant strains and the adverse impact on the protective effect of 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 first aspect of the present invention provides a method for improving the ECD antigen immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strains A novel method for obtaining a stable trimeric form of a pre-fusion conformation by constructing an ECD antigen comprising the amino acid sequence shown in SEQ ID No: 8, or an immunogenic fragment and/or an immunogenic variant thereof ECD.

In one embodiment, the mutant strain contains A67V, H69del, V70del, T95l, G142D, V143del, Y144del, Y145del, N211del, L212l, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K , E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F are high-risk mutant strains with at least one mutation;

Preferably, the mutant strain is BA.1.

In one embodiment, the ECD antigen and an adjuvant are co-administered to the subject, and the adjuvant is selected from the group consisting of aluminum adjuvants, oil-emulsion adjuvants, Toll-like receptor (TLR) agonists, combinations of immunopotentiators, microbial adjuvants one or more of propolis adjuvant, levamisole adjuvant, liposome adjuvant, traditional Chinese medicine adjuvant and small peptide adjuvant;

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 immune boosters include QS-21 and/or MPL.

The second aspect of the present invention provides a method for improving the immunogenicity of the SARS-CoV-2 mutant strain ECD antigen immunogenicity/antigen trimer stability, the method comprises the amino acid sequence shown in SEQ ID No: 8 by constructing the code, or Polynucleotides of immunogenic fragments and/or immunogenic variants thereof, thereby expressing the trimeric form of the ECD in a stable prefusion conformation.

Preferably, the mutant strain is BA.1.

In one embodiment, the method comprises constructing a polynucleotide comprising the nucleotide sequence shown in SEQ ID No: 7 or a fragment thereof.

The third aspect of the present invention provides a SARS-CoV-2 mutant strain ECD immunogenic protein/peptide with improved immunogenicity/antigen trimer stability, the immunogenic protein/peptide comprising SEQ ID No: 8 The amino acid sequence shown, or immunogenic fragments and/or immunogenic variants thereof, the ECD immunogenic protein/peptide is in the form of a trimer in a stable pre-fusion conformation.

In one embodiment, the mutant strain contains A67V, H69del, V70del, T95l, G142D, V143del, Y144del, Y145del, N211del, L212l, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K , E484A, High-risk mutant strains with at least one mutation among Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F;

Preferably, the mutant strain is BA.1.

The fourth aspect of the present invention provides a polynucleotide encoding the above-mentioned immunogenic protein/peptide.

In one embodiment, the polynucleotide comprises the nucleotide sequence shown in SEQ ID No: 7.

The fifth aspect of the present invention provides an immunogenic composition comprising:

an immunogenic protein/peptide as described above, or

the aforementioned polynucleotides, and

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

Optionally, an adjuvant is included.

In one embodiment, the immunogenic composition further comprises amino acid sequences shown in SEQ ID No: 16, SEQ ID No: 20, SEQ ID No: 28, or immunogenic fragments thereof and/or immunogenic Variants.

In one embodiment, the immunogenic composition further comprises amino acid sequences encoding SEQ ID No: 16, SEQ ID No: 20, SEQ ID No: 28, or immunogenic fragments and/or immunogens thereof Nucleotide sequence of the sex variant.

In one embodiment, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID No: 16, SEQ ID No: 20, and SEQ ID No: 28 are SEQ ID No: 15, SEQ ID No: 19, SEQ ID No: 19, and SEQ ID No: 28, respectively. ID No: the nucleotide sequence shown in 27.

In one embodiment, the adjuvant is 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 One or more of the peptide adjuvants.

Preferably, the oil-emulsion adjuvant contains squalene;

Combinations of immune boosters include QS-21 and/or MPL.

The sixth aspect of the present invention provides the use of the above-mentioned immunogenic protein/peptide, polynucleotide and immunogenic composition for preventing or treating diseases caused by mutant SARS-CoV-2 strains.

In one embodiment, the mutant strain is a high-risk mutant strain;

Preferably, mutant strains are containing L18F, T19L, T19R, L24DEL, P25DEL, P26DEL, A27S, A67V, H68DEL, H69DEL, V70DEL, D80A, T95L, G143DEL, Y145DEL, Y145DEL, E156G, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15 7DEL, R158DEL, N211DEL, L212l, V213G, ins214EPE, D215G, L242del, A243del, L244del, R246l, G339D, R346K, S371F, S371L, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, L 452R, S477N, T478K, E484A, E484K, E484Q, F486V, Q493R, G496S, Q498R, N501Y, Y505H, T547K, A570D, D614G, H655Y, N679K, P681H, P681R, A701V, T716l, N764K, D796Y, N856K, D950N, Q9 54H, N969K, L981F, S982A and D1118H High-risk mutant strains with at least any mutation in;

In one embodiment, the strain is selected from D614G mutant strain, Beta strain, Alpha strain, Delta strain, Gamma strain, Epsilon strain, BA.1 strain, BA.1.1 strain, BA.2 At least one of strains, BA.2.12.1 strains, BA.3 strains and/or BA.4/5 strains;

Preferably, the strain comprises Alpha strain, Beta strain, Delta strain, BA.1 strain, BA.1.1 strain, BA.2 strain, BA.2.12.1 strain, BA.3 strain and/or at least one of the BA.4/5 strains.

The seventh aspect of the present invention provides the use of the above-mentioned immunogenic protein/peptide, polynucleotide and immunogenic composition in the preparation of vaccines or medicines for preventing or treating diseases caused by mutant SARS-CoV-2 strains.

In one embodiment, the mutant strain is a high-risk mutant strain;

In one embodiment, the strain is selected from D614G mutant strain, Alpha strain, Beta strain, Delta strain, Gamma strain, Epsilon strain, BA.1 strain, BA.1.1 strain, BA.2 At least one of strains, BA.2.12.1 strains, BA.3 strains and/or BA.4/5 strains;

More preferably, the strain is selected from Alpha strain, Beta strain, Delta strain, BA.1 strain, BA.1.1 strain, BA.2 strain, BA.2.12.1 strain, BA.3 strain and/or at least one of the BA.4/5 strains.

Description of drawings

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

Figure 2 is the analysis of the purity of TM41 protein, wherein (A) is a representative spectrum of non-reducing SDS-PAGE; (B) is a representative spectrum of SEC-HPLC.

Figure 3 shows the detection results of serum antibody titers (GeoMean±SD) after immunizing C57BL/6 mice with TM41 single-component and multi-component vaccines.

Figure 4 shows the neutralization titer detection results (GeoMean±SD) of different pseudoviruses neutralized in serum after immunization of C57BL/6 mice with TM41 single-component and multi-component vaccines for 2 days and 7 days.

Fig. 5 shows different doses of TM22+TM23+TM28+TM41 four-component vaccine to immunize C57BL/6 mice for 2 days and 7 days after serum neutralizes the titer detection (GeoMean±SD) of different mutant strains of pseudovirus.

Fig. 6 shows TM22+TM23 two-component vaccine and TM22+TM23+TM28+TM41 four-component vaccine immunization C57BL/6 mouse 2 immunizations after 7 days serum neutralizes different variant strain pseudovirus titer detection results (GeoMean±SD ).

Fig. 7 shows C57BL/6 mouse 2 immunization 7 days and 3 immunization 7 days serum and Omicron (BA.1) pseudovirus titer detection result (GeoMean ± SD); 2M7D represents 2 immunizations 7 days; 3 7 days after exemption.

Figure 8 shows the titer detection results of TM22+TM23+TM28+TM41 four-component vaccine and single-component/two-component vaccine immunization of C57BL/6 mice for 2 days and 14 days after serum neutralization of different mutant pseudoviruses.

Figure 9 shows the detection results of cellular immune responses induced by different vaccine antigens, wherein (A) is the result of the number of IFN-positive cells; (B) is the result of the number of IL-4 positive cells; (C) is the result of the number of CD137+CD134+ double positives The results of the proportion of CD4 T lymphocytes; (D) is the result of the proportion of CD137+CD69+ double positive CD8 T lymphocytes.

Figure 10 (AD) shows the comparison of the neutralization titers of the serum to each subtype of Omicron (BA.1, BA.2, BA.3, BA.4/5) pseudoviruses after booster immunization. [a indicates the fold change compared with TM8 two-shot immunization (2 immunizations for 7 days); b indicates the fold change compared with TM8 booster immunization].

Figure 11 (A-C) shows the comparison of neutralizing titers of sera to Alpha, Beta, and Delta pseudoviruses after 1 shot of booster immunization respectively. [a indicates the fold change compared with TM8 two-shot immunization (2 immunizations for 7 days); b indicates the fold change compared with TM8 booster immunization].

Figure 12 (A-D) respectively shows the comparison of the neutralization titer of serum to each subtype of Omicron (BA.1, BA.2, BA.3, BA.4/5) pseudoviruses after 2 injections of booster immunization. [a indicates the fold change compared with TM8 two-shot immunization (2 immunizations for 7 days); b indicates the fold change compared with TM8 booster immunization].

In the icons of the drawings, Omicron (B.1.1.529) and Omicron both refer to Omicron (BA.1).

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". A "hapten" refers to 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).

The term "humoral immune response" is an antibody-mediated immune response and involves the introduction and production of antibodies that recognize and bind with a certain affinity to the antigen in the immunogenic composition of the invention, a "cell-mediated immune response" is an immune response mediated by T cells 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 "immunogenic fragments" and "immunogenic variants" of the present invention are 99%, 98%, 97%, 95%, 90% identical to the corresponding (the former derived from the latter) "immunogenic protein/peptide" sequence identity.

The SCTV01E 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 natural spike protein of SARS-CoV-2 is a trimeric structure. During its production and infection function, it is easily cleaved by proteases in the Golgi apparatus and on the cell surface due to the RRAR site between S1 and S2 After opening, S1 falls off, and the S2 structure changes from prefusion conformation to postfusion conformation, thus completing the membrane fusion process[20]. In order to obtain the ECD trimer of the stable pre-fusion conformation, the present invention carried out the following three-part transformation on the basis of the S protein of different strain variants (Table 1 and Fig. 1):

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 S1 part intact 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 SCTV01E recombinant protein vaccine, that is, the amino acid sequence from 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 pre-fusion conformation of the spike protein is unstable, and the effective induction of neutralizing antibodies requires a stable pre-fusion conformation, which has been confirmed in RSV and HIV-1 vaccine studies ^{[21 , 22]} . Most of the new crown vaccines currently on the market or in the clinical stage focus on the part of the spike protein that plays an important role in virus infection, so how to ensure its stability in the pre-fusion conformation has become a concern. In the current marketed vaccines, the S-2P (that is, amino acid mutations at positions 986 and 987 to proline) modification scheme is commonly used ^[23-25] . In order to further improve the conformational stability of ECD before fusion, so as to improve its expression level and product stability in CHO recombinant cells, so as to make it convenient for storage and transportation while reducing production costs, the present invention introduces the SCTV01E recombinant protein vaccine HexaPro mutations that can effectively improve 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 to proline) ^[26] . These mutation sites are all located at the N-terminal or Loop region of the α-helix in S2, and after being mutated to the proline (P) type with this secondary structure tendency, it can effectively reduce the allosteric tendency of S2 and thus stabilize the fusion of S2 pre-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. T4 foldon has been used in RSV vaccine candidates, and proved to be safe in Phase I clinical studies ^[27] .

After using the recombinant S-ECD trimer protein antigen recombined into the expression vector after the above transformation, and performing routine purity and stability analysis on the expressed recombinant S-ECD trimer protein, the corresponding trimer was prepared Protein, that is, in the previous invention (recorded in PCT/CN2022/095609 and PCT/CN2022/107213) D614G mutant strain S-Trimer-TM8 protein (hereinafter referred to as TM8), Alpha mutant strain S-Trimer-TM22 protein (hereinafter referred to as TM22) , Beta mutant strain S-Trimer-TM23 protein (hereinafter referred to as TM23), Delta mutant strain S-Trimer-TM28 protein (hereinafter referred to as TM28) and BA.1 mutant strain S-Trimer-TM41 protein of the present invention (hereinafter referred to as TM41, Table 1 outlines its molecular modification scheme).

In the previous invention, the prepared D614G variant strain TM8 protein vaccine was used to immunize mice for immunological determination, the immunological determination of Beta variant strain TM23 protein vaccine in cynomolgus monkeys and the Alpha variant strain TM22 protein vaccine in mice Immunological assays all show that these three vaccines prepared by the present invention can produce antibody immune responses of sufficient titer in experimental animals; It is also suggested in the immunological evaluation that the two-component vaccine of the present invention has higher and similar neutralizing titers to different strains, so it has better broad-spectrum neutralization ability than the single-component vaccine. The neutralizing titer for different mutant strains is much higher than that of the convalescent serum for the early epidemic strain (its genome sequence: GenBank Accession No.NC_045512).

The four-component vaccine (TM22+TM23+TM28+TM41) of the present invention has more broad-spectrum neutralizing activity to mutant strains such as Alpha, Beta, Delta and Omicron, but keeps the same single-component vaccine and two-component vaccine. A similar high level of T cell immune response is expected to produce cross-protection against a variety of mutant strains (Table 2 records the S protein mutations of related SARS-CoV-2 mutant strains) and improve the protection rate against mutant strain infection .

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.

Table 1 Molecular structure design and modification of SCTV01E vaccine

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

Note: The sequence is from GISAID (https://gisaid.org/)

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 Construction of S-ECD trimer protein (S-Trimer-TM41) expression vector based on BA.1 (B.1.1.529.1) sequence (EPI_ISL_6640917)

TM41 contains a 3699bp gene fragment, and the TM41 gene fragment was obtained by overlapping PCR from the template pCMV3-CoV2-B.1.1.529 and pD2535nt-CoV2-S-ECDTM8-T4F-trimer. Constructed into the pD2535nt-HDP stable strain expression vector digested with Xba I+Asc I by the In-fusion method to obtain the pD2535nt-CoV2-S-ECDTM41-T4F-trimer expression vector.

Amplification primer

1.2 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

Put the above-mentioned purified recombinant S-ECD trimer protein stock solution in a buffer solution containing 1.7mM citric acid, 8mM sodium citrate, 300mM sodium chloride, 0.3g/kg polysorbate 80, pH7.0±0.2 , 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 molecular exclusion high performance liquid chromatography (siza-exclusion) High performance liquid chromatograph (SEC-HPLC) was used to analyze its trimer content, and dynamic light scattering (dynamic light scattering, DLS) was used to detect its morphological characteristics.

SDS-PAGE specific operation steps: (1) SDS-PAGE gel preparation: 3.9% stacking gel, 7.5% separating gel; (2) Samples were boiled at 100°C for 2 minutes, centrifuged and loaded with 8 samples-; (3) Coomassie brilliant blue staining After bleaching. 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 NaH2P04, 100mM Arginine, pH 6.5, 0.01% isopropanol (IPA); (3) The sample volume is 80μg; (3) The detection wavelength is 280nM, the analysis time is 35min, and the flow rate is 0.15mL/min.

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 recombinant TM41 protein has a homotrimeric structure due to its non-covalent hydrophobic interaction. After non-reducing SDS-PAGE treatment, it becomes a monomer molecule with a molecular weight of about 148KDa (Figure 2), and the purity is 99.0%. %, the average molecular weight of its main peak is 512KDa; the dynamic light scattering results show that the average molecular radius of TM41 trimeric protein is 8.8nm (Table 3).

Table 3 Recombinant S-ECD trimer purity analysis

2.2 Stability evaluation of recombinant S-ECD trimer protein

Recombinant TM41 trimer protein was stored at 37°C for 2 weeks (37T2W), stored at -80°C for 8 hours, and then transferred to 25°C for 0.5h (F/T), and repeated freezing and thawing was carried out 4 times. SDS-PAGE, SEC-HPLC analysis of its trimer content changes, the data see Table 4.

The results are shown in Table 4. After the recombinant TM41 trimer protein was accelerated at 37°C for 2 weeks and after 5 repeated freeze-thaw cycles, the non-reducing SDS-PAGE purity and SEC-HPLC trimer content were both above 95.0%. The change is within 2.0%, there is no significant increase in aggregates and fragments, and it shows good thermal acceleration stability and freeze-thaw stability.

Table 4 Recombinant S-ECD trimer protein stability evaluation

Embodiment 3: TM41 single-component vaccine and multi-component vaccine in Immunological evaluation of mice

3.1 Vaccine preparation and immunization scheme

For the expression and purification of TM22 and TM23 trimer proteins, see PCT/CN2022/095609 "A Method for Improving ECD Antigen Immunogenicity/Stability of Antigen Trimers of SARS-CoV-2 Mutant Strain" (herein, the full text is introduced) . In this patent, the applicant elaborated that the two-component vaccine composed of TM22+TM23 has better broad-spectrum neutralization ability than the single-component vaccine of TM22 and TM23. For the expression and purification of the TM28 trimer protein, see PCT/CN2022/107213 "Preparation and Application of a Recombinant Multi-Component Trimeric Protein Vaccine with Inducible Broad-Spectrum Neutralization Activity" (introduced in its entirety here). In the patent application, the applicant elaborated that the trivalent vaccine composed of TM22+TM23+TM28 has better broad-spectrum neutralization ability than the single-component vaccine. In order to further broaden the broad-spectrum neutralizing effect of the vaccine, especially for the neutralizing effect of the Omicron variant, the applicant added TM41 to the TM22+TM23+TM28 trivalent vaccine to form a four-component vaccine.

According to the final immune dose (Table 5), the purified TM22, TM23, TM28 and TM41 trimeric proteins were pre-diluted with PBS and mixed with MF59 (8×, source: Shenzhou Cell Engineering Co., Ltd., the same below) in equal volumes to prepare Single or multi-component vaccine samples.

Table 5 Summary of immunization grouping information

3.2 Immunization of mice

6-8 week old female C57BL/6 mice (source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., weighing 18-20 g) were intramuscularly injected with 0.1 mL of vaccine samples containing MF59 adjuvant. A total of 3 immunizations were carried out with an interval of 14 days. Orbital blood was collected 7 days after the second immunization (7 days after the second immunization) and 7 days after the third immunization, and the serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immune analysis.

3.3 Determination of antibody titer of mouse immune serum

The immune serum of mice immunized with one-component vaccine was coated with 5 μg/mL of TM41 protein, the immune serum of mice immunized with two-component vaccine was coated with 5 μg/mL of TM22 and TM23 proteins (1:1), and the immune serum of mice immunized with two-component vaccine was coated with 5 μg/mL of TM22 and TM23 proteins (1:1), TM22, TM23, TM28 and TM41 proteins (1:1:1:1) were coated with 5 μg/mL of mouse immune serum, 100 μL/well was coated on a 96-well plate, and coated overnight at 2-8°C. After the microplate was washed and patted dry, 320 μL/well of blocking solution containing 2% BSA was added to block at room temperature for more than 1 h. Use the TBST sample diluent containing 0.1% BSA to serially dilute the single-component or multi-component vaccine mouse immune serum (such as 8000×, 16000×, 32000×, 64000×, 128000×, 256000×, 512000×, etc.) , Serum from unimmunized mice diluted in the same gradient 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, read the OD ₄₅₀ with a microplate reader after the termination of 2M H ₂ SO 4 , and calculate the immune antibody titer. Antibody titer = negative serum OD ₄₅₀ × maximum dilution factor of 2.1.

The results of the total IgG antibody titer in the serum of mice 7 days after the second immunization are shown in Figure 3. Compared with the blank control group and the adjuvant control group, TM22+TM23 two-component vaccine antigen, different doses of TM41 single-component vaccine antigen and TM22 +TM23+TM28+TM41 four-component vaccine antigen immunization groups all induced higher levels of total IgG antibody titers in mouse serum. Among them, the total IgG antibody titer induced by the TM22+TM23 two-component vaccine antigen was 960000, while the TM22+TM23+TM28+TM41 four-component vaccine antigen immunization group (0.5+0.5+0.5+1.5μg) induced the highest total IgG antibody titer. IgG antibody titer (1024000). The total IgG antibody titers induced by different doses of TM41 single-component vaccine antigen (0.25 μg/dose, 0.5 μg/dose and 1 μg/dose) showed a dose-effect relationship, and the antibody titers were 256,000, 512,000, and 576,000, respectively. The total IgG antibody titers induced by different doses of TM22+TM23+TM28+TM41 four-component vaccine antigen immunization group were higher than that of TM41 single-component vaccine.

3.4 Determination of neutralizing titer of mouse immune serum to different mutant strains

Add 50 μL/well of 2-immune 7-day immune serum or 3-immune 7-day immune serum with different dilutions into 96-well plate, and then add 100-200 TCID ₅₀ of D614G, Alpha (B.1.1.7), Beta (B .1.351), Delta (B.1.617.2) and Omicron different subtype mutant strain pseudoviruses (pseudoviruses are replication-deficient vesicular stomatitis viruses that replace the VSV-G protein gene in the viral genome with the luciferase reporter gene (i.e. VSVΔG-Luc-G) as a carrier, amplified and prepared in a cell line expressing Spike and its mutant proteins, prepared by Shenzhou Cell Engineering Co., Ltd., the same below), mixed and placed at 37 ° C, 5% Incubate for 1 h in a CO ₂ incubator. 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%, IC50 was calculated according to the Reed-Muench formula, which was the neutralization titer NAT ₅₀ .

3.4.1 Determination of the neutralizing titer of TM41 single-component and multi-component vaccine immune sera to different mutant strains

Detection of TM41 single-component and four-component vaccines TM22+TM23+TM28+TM41 immunized C57BL/6 mice for 2 days and 7 days after serum neutralization of different mutant strains Alpha(B.1.1.7), Beta(B.1.351), Delta (B.1.617.2) and BA.1 (B.1.1.529.1) pseudovirus neutralization potency. The TM41 single-component vaccine at different doses can induce the specific neutralizing antibodies against the BA.1 variant strain, and 1 μg of the single-component vaccine TM41 can induce the highest anti-BA.1 variant strain in C57BL/6J mice Neutralizing activity, the neutralizing antibody titer was 2730, which reached the saturation dose in this experiment. However, the TM41 single-component vaccine has no neutralizing activity against the Alpha, Beta and Delta mutant strains, and the neutralizing antibody titer detection values are all 60, which is lower than the detection limit. The TM22+TM23+TM28+TM41 four-component vaccines in different dosage groups had strong neutralizing activity against BA. 1 The detection value of the neutralizing antibody titer of the variant strain was 618-2730. The neutralizing activity of the TM22+TM23+TM28+TM41 four-component vaccine against the BA.1 mutant strain is comparable to that of the TM41 single-component vaccine, and it also has higher neutralizing activity against the Alpha, Beta, Delta and other mutant strains, indicating that Compared with the TM41 single-component vaccine, the TM22+TM23+TM28+TM41 four-component vaccine has a broader spectrum of neutralizing activity against different variants of SARS-CoV-2 (Figure 4).

3.4.2 Determination of the neutralization titer of different doses of TM22+TM23+TM28+TM41 four-component vaccine immune serum to different mutant strains

Detect different doses of TM22+TM23+TM28+TM41 four-component vaccine to immunize C57BL/6 mice for 2 days and 7 days to neutralize different mutant strains Alpha(B.1.1.7), Beta(B.1.351), Delta(B. 1.617.2) and BA.1 (B.1.1.529.1) pseudovirus neutralization titers. Different doses of TM22+TM23+TM28+TM41 four-component vaccines can induce higher levels of neutralizing antibodies against Alpha, Beta and Delta variants. Compared with the above three neutralizing antibodies, the four-component vaccines induced BA .1 The overall level of neutralizing antibodies is low. As the dose of TM41 increased, the neutralizing activity against BA.1 mutant strains tended to increase, showing a dose-effect relationship (Fig. 5).

3.4.3 Determination of the neutralizing potency of TM22+TM23 two-component vaccine and different doses of TM22+TM23+TM28+TM41 four-component vaccine immune serum to different mutant strains

Detection of TM22+TM23 two-component vaccine and different doses of TM22+TM23+TM28+TM41 four-component vaccine immunized C57BL/6 mice for 2 days and 7 days after serum neutralization of different mutant strains Alpha(B.1.1.7), Beta( B.1.351), Delta (B.1.617.2) and BA.1 (B.1.1.529.1) pseudovirus titers. The specific neutralizing antibody titers of the TM22+TM23 two-component vaccine group against the Alpha, Beta, Delta and BA.1 mutant strains were 4598, 4972, 1384 and 247, respectively. Higher specific neutralizing antibody titers against Alpha, Beta, and Delta were generated, but the protective effect on BA.1 mutant strains was relatively weak. The neutralizing antibody titers produced by the TM22+TM23+TM28+TM41 four-component vaccine at different doses against the Alpha and Beta mutant strains were comparable to those of the two-component vaccines, while the neutralizing antibody titers against the Delta and BA.1 mutant strains than two-component vaccines. Among them, the low-dose group of the four-component vaccine induced 5.4 times and 1.9 times the neutralizing antibodies against the Delta and BA.1 variants than the two-component vaccine, and the medium dose of the four-component vaccine induced neutralizing antibodies against the Delta and BA.1 variants. The neutralizing antibodies against the Delta and BA.1 variants induced by high doses of the four-component vaccine were 4.2 and 5.9 times higher than those of the two-component vaccine (Figure 6). It shows that the protective effect of TM22+TM23+TM28+TM41 four-component vaccine is better than that of TM22+TM23 two-component vaccine when protecting Delta and BA.1 mutant strain infection.

3.4.4 Determination of the neutralization titer of vaccine immune sera with different immunization schemes to Omicron strain

Detect the neutralization titer of BA.1 (B.1.1.529.1) pseudovirus in serum of C57BL/6 mice for 7 days after 2 immunization and 7 days after 3 immunization. Serological test results showed that, compared with the BA.1 neutralizing antibody titers in the serum after the 2nd immunization and 7 days after the 3rd immunization and 7 days, the BA.1 neutralizing antibody titers of all vaccine immunization groups showed a rising trend, TM22+TM23 Two-component vaccine increased by 0.9 times, TM41 (0.25, 0.5, 1 and 2μg) increased by 7.8, 3.9, 1.2 and 0.8 times, respectively, TM22+TM23+TM28+TM41 four-component vaccine (0.25μg/valent, 0.5μg/valent, 1μg/valent and 2μg/valent) increased by 4.9, 4.2, 1.6 and 2.6 times, respectively. Four-component vaccine 0.5+0.5+0.5+1 μg and 0.5+0.5+0.5+1.5 μg increased by 2.6 times and 2.4 times respectively (Figure 7).

3.4.5 Determination of the neutralizing titer of TM22+TM23+TM28+TM41 four-component vaccine and single-component/two-component vaccine immune serum to different mutant strains

Detection of TM22+TM23+TM28+TM41 four-component vaccine and single-component/two-component vaccine immunization of C57BL/6 mice for 14 days after 2 immunizations to neutralize different mutant strains (D614G strain, Alpha strain, Beta strain, Delta strain, BA.1 strain, BA.1.1 strain, BA.2 strain, BA.2.12.1 strain, BA.3 strain and BA.4/5 strain) pseudovirus titer. Comparing the neutralizing activity of the four-component vaccine TM22+TM23+TM28+TM41 with the TM8, TM41 single-component vaccine and the two-component vaccine TM22+TM23 against different new coronavirus mutant strains pseudoviruses, the test results are shown in Figure 8A. Compared with TM8 single-component vaccine, the four-component vaccine TM22+TM23+TM28+TM41 can significantly improve the response to Beta, Delta, Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4/5 variation Strain neutralizing activity. As shown in Figure 8B, compared with the TM41 single-component vaccine, the four-component vaccine TM22+TM23+TM28+TM41 can significantly improve the neutralizing activity against D614G, Alpha, Beta and Delta mutant strains. As shown in Figure 8C, compared with the two-component vaccine TM22+TM23, the four-component vaccine TM22+TM23+TM28+TM41 can significantly improve the variation of Delta, Omicron BA.1, BA.1.1, BA.2, and BA.3 Strain neutralizing activity. As shown in Figure 8D, after increasing the vaccine immunization dose to 6 micrograms per dose, compared with the TM41 single-component vaccine, the four-component vaccine TM22+TM23+TM28+TM41 can significantly improve the effect on Omicron BA.2.12.1 and BA.4/ 5 Neutralizing activity of mutant strains.

3.5 Detection of vaccine-induced T cell immune response

ELISpot method for detection of T cell immunity: isolate mouse splenocytes, inoculate 100 μL/well of mouse splenocytes on pre-treated ELISpot well plates (source: Mabtech, the same below), and the cell inoculation density is 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 GraphPad Prism software was used for data statistics.

Detect activated T cell subsets by flow cytometry: Grind the spleen into a single cell suspension, use wild-type (original strain: genome sequence: GenBank Accession No.NC_045512) polypeptide library and Omicron (BA.1) polypeptide library Stimulate splenocytes immunized with different vaccine antigens for 20 hours in a 37°C 5% CO ₂ incubator. After the stimulation, wash the cells with PBS, centrifuge at 1000rpm for 5min and discard the supernatant. According to the detection requirements, use BV510 anti- mouse CD3e, CD4 Antibody (FITC), Rabbit Mab, CD8a Antibody (APC), Rabbit Mab, BV650 Hamster Anti-Mouse CD69, PE Rat Anti-Mouse CD137, Brilliant Violet 421TM anti-mouse CD134 (OX-40) corresponding antibody Spleen cells were stained at 4°C in the dark for 20 minutes, and detected by flow cytometry after staining.

The results of ELISpot detection of vaccine-induced T cell immune responses showed that the level of Th1 cell immunity (the number of IFN Y positive cells) induced by the TM41 single-component vaccine antigen was comparable to that induced by the TM22+TM23 two-component vaccine antigen, which was comparable to that of the above two vaccines. Compared with that, the four-component vaccine antigen of TM22+TM23+TM28+TM41 can induce a higher level of Th1 cell immune response, showing its superiority (Figure 9A). For vaccine-induced Th2 cell immune responses, the number of IL-4 positive cells induced by TM41 single-component, TM22+TM23 two-component and TM22+TM23+TM28+TM41 four-component vaccine antigens was comparable, and the vaccine-induced Th2 cells The immune response was not significantly different between the groups (Fig. 9B). After the three groups of different vaccines were immunized, the spleen cells of the mice were stimulated with wild-type and BA.1 antigen peptides respectively, and the activated CD4 ⁺ and CD8 ⁺ T cells were induced higher than the levels of the blank and adjuvant control groups, and There were no significant differences between the three live vaccines. The wild-type and BA.1 antigen peptides have similar T cell activation stimulation levels, indicating that there are conserved T cell epitopes among different strains (Fig. 9C-D).

Example 4: Immunological evaluation of TM22+TM23+TM28+TM41 four-component vaccine in Naive mice

4.1 Immunization scheme and mouse immunization

Refer to Example 3.1 to prepare TM8 single-component vaccine, TM22+TM23 two-component vaccine and TM22+TM23+TM28+TM41 four-component vaccine.

About 6 weeks old, C57BL/6J female mice (source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were immunized with 100 μL TM8 single-component vaccine antigen containing MF59 adjuvant by intramuscular injection on day 0 and day 14 respectively ( 1 μg/dose), and the serum immunoassay was carried out in the orbit on the 2nd and 7th day. On the 70th day and the 182nd day, the TM22+TM23+TM28+TM41 four-component vaccine, the TM22+TM23 two-component vaccine, and the TM8 single-component vaccine were boosted with the same immunization method (antigen dose: 1 μg/dose, Among them, the distribution ratio of each group of TM22+TM23 is TM22:TM23=1:1, and the distribution ratio of each group of TM22+TM23+TM28+TM41 is TM22:TM23:TM28:TM41=1:1:1:3; MF59 adjuvant dosage: 2 mg/dose), and 8 mice in each group were immunized 4 times in total. Blood was collected 7 days after the single-shot and double-shot booster immunizations for serological immune analysis.

4.2 Determination of neutralizing titer of mouse immune serum

4.2.1 Determination of the neutralizing titer of the immune serum to the mutant strain after a booster immunization in mice

With reference to Example 3.4, detect the neutralizing titer of mouse immune serum to Alpha, Beta, Delta, each subtype of Omicron (BA.1, BA.2, BA.3, BA.4/5) pseudovirus. Geometric mean neutralization titers of Omicron BA.1, BA.2, BA.3, BA.4/5 pseudoviruses induced by TM22+TM23+TM28+TM41 four-component vaccine single-shot booster immunization (3 immunizations and 7 days) The values were 1293, 1178, 803 and 722, respectively, which were 15.0 times, 9.1 times, 10.7 times and 10.6 times of the neutralization titer of the TM8 single-component vaccine 7 days after the second immunization, and 2.9 times of the TM8 single-component vaccine booster immunization group. times, 3.7 times, 2.3 times and 3.0 times, which were 2.9 times, 3.8 times, 4.1 times and 3.9 times that of the TM22+TM23 two-component vaccine booster group (Fig. 10A-D); the neutralization titer to the Delta strain was 22298 , which is 5.2 times the neutralization titer of TM8 single-component vaccine 7 days after the second immunization, 1.6 times that of the single-component vaccine booster immunization group, and 1.6 times that of the two-component vaccine booster immunization group; in addition, TM22+TM23+TM28 +TM41 four-component vaccine booster immunization can induce high levels of neutralizing activities of the Alpha and Beta variants, which are 21257 and 12898, respectively (Fig. 11A-C). It is suggested that the four-component vaccine has a broader neutralizing activity against SARS-CoV-2 mutants Alpha, Beta, Delta and Omicron (BA.1, BA.2, BA.3, BA.4/5), and is superior to Booster immunization with TM8 single-component vaccine or TM22+TM23 two-component vaccine.

4.2.2 Determination of the neutralizing titer of the immune serum to the mutant strain after two doses of booster immunization in mice

The geometric mean neutralization titers of Omicron BA.1, BA.2, BA.3 and BA.4/5 induced by two injections of TM22+TM23+TM28+TM41 four-component vaccine after booster immunization (4 days and 7 days) were respectively 14872, 6897, 8768 and 1136, which were 172.9 times, 53.5 times, 116.9 and 16.7 times the neutralization titers of the TM8 single-component vaccine 7 days after the second immunization, and 22.6 times and 17.4 times the TM8 single-component vaccine booster immunization group. times, 32.5 times and 2.8 times, which are 20.7 times, 6.7 times, 20.8 times and 1.7 times of the TM22+TM23 two-component vaccine booster group (Figure 12). It is suggested that two booster immunizations of TM22+TM23+TM28+TM41 four-component vaccine can induce a high level of neutralizing activity of 0micron mutant strains (BA.1, BA.2, BA.3, BA.4/5), which is better than TM8 One-component vaccine booster immunization or two-component vaccine booster immunization.

In summary, compared with the single-component vaccine and the two-component vaccine, the four-component vaccine has a broad-spectrum neutralization ability against different mutant strains, but maintains a similar high The level of T cell immune response is expected to produce cross-protection ability 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.

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Claims

A method for improving the immunogenicity of the SARS-CoV-2 mutant strain ECD antigen immunogenicity/antigen trimer stability, characterized in that the method comprises the amino acid sequence shown in SEQ ID No: 8, or its immunogen ECD antigens of sexual fragments and/or immunogenic variants to obtain a stable trimeric form of ECD in a prefusion conformation;

Preferably, the mutant strain contains A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T4 78K, E484A, High-risk mutant strains with at least one mutation among Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F;

Preferably, the mutant strain is BA.1;

Preferably, the ECD antigen and an adjuvant are co-administered to the subject, and the adjuvant is 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 One or more of peptide adjuvants;

Preferably, the oil-emulsion adjuvant contains squalene;

Preferably, the Toll-like receptor (TLR) agonist comprises CpG or monophosphoryl lipid A (MPL) adsorbed on an aluminum salt;

Preferably, the combination of immunopotentiators comprises QS-21 and/or MPL.
A method for improving the immunogenicity of the SARS-CoV-2 mutant strain ECD antigen immunogenicity/antigen trimer stability, is characterized in that, the method comprises the aminoacid sequence shown in SEQ ID No: 8 by constructing coding, or its immune polynucleotides of the original fragment and/or the immunogenic variant, thereby expressing the trimeric form of the ECD in a stable prefusion conformation;

Preferably, the mutant strain contains A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T4 78K, E484A, High-risk mutant strains with at least one mutation among Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F;

More preferably, the mutant strain is BA.1;

Most preferably, the method comprises constructing a polynucleotide comprising the nucleotide sequence shown in SEQ ID No: 7 or a fragment thereof.
A SARS-CoV-2 mutant strain ECD immunogenic protein/peptide with improved immunogenicity/antigen trimer stability, characterized in that the immunogenic protein/peptide comprises SEQ ID No: 8 Amino acid sequences, or immunogenic fragments and/or immunogenic variants thereof, of the ECD immunogenic protein/peptide in the form of a trimer in a stable prefusion conformation;

Preferably, the mutant strain contains A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T4 78K, E484A, High-risk mutant strains with at least one mutation among Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F;

Preferably, the mutant strain is BA.1.
A polynucleotide encoding the immunogenic protein/peptide of claim 3;

Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID No: 7.
An immunogenic composition, characterized in that the immunogenic composition comprises:

The immunogenic protein/peptide of claim 3, or

The polynucleotide of claim 4, and

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

Optionally, an adjuvant is included.
5. The immunogenic composition of claim 5, wherein the immunogenic composition further comprises:

The amino acid sequence set forth in SEQ ID No: 16, SEQ ID No: 20, SEQ ID No: 28, or immunogenic fragments and/or immunogenic variants thereof, or

A nucleotide sequence encoding the amino acid sequence shown in SEQ ID No: 16, SEQ ID No: 20, SEQ ID No: 28, or an immunogenic fragment and/or immunogenic variant thereof,

Preferably, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID No: 16, SEQ ID No: 20, and SEQ ID No: 28 are respectively SEQ ID No: 15, SEQ ID No: 19, and SEQ ID No: The nucleotide sequence shown in 27.
5. The immunogenic composition of claim 5, wherein 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;

Preferably, the Toll-like receptor (TLR) agonist comprises CpG or monophosphoryl lipid A (MPL) adsorbed on an aluminum salt;

Preferably, the combination of immunopotentiators comprises QS-21 and/or MPL.
The immunogenic protein/peptide of claim 3, the polynucleotide of claim 4, and the immunogenic composition of claim 5 or 6 are used for preventing or treating SARS-CoV-2 mutant strains causing The use of the disease, preferably, the mutant strain is a high-risk mutant strain;

Preferably, mutant strains are containing L18F, T19i, T19R, L24DEL, P25DEL, P26DEL, A27S, A67V, H68DEL, H69DEL, V70DEL, D80A, T95i, G142D, V144DEL, Y145DEL, E156G, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15 7DEL, R158DEL, N211DEL, L212I, V213G, ins214EPE, D215G, L242del, A243del, L244del, R246I, G339D, R346K, S371F, S371L, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, L 452R, S477N, T478K, E484A, E484K, E484Q, F486V, Q493R, G496S, Q498R, N501Y, Y505H, T547K, A570D, D614G, H655Y, N679K, P681H, P681R, A701V, T716I, N764K, D796Y, N856K, D950N, Q9 54H, N969K, L981F, S982A and D1118H High-risk mutant strains with at least any mutation in;

Preferably, the strain is selected from D614G mutant strain, Beta strain, Alpha strain, Delta strain, Gamma strain, Epsilon strain, BA.1 strain, BA.1.1 strain, BA.2 strain, At least one of BA.2.12.1 strain, BA.3 strain and/or BA.4/5 strain;

More preferably, the strain comprises Alpha strain, Beta strain, Delta strain, BA.1 strain, BA.1.1 strain, BA.2 strain, BA.2.12.1 strain, BA.3 strain strain and/or at least one of the BA.4/5 strains.
The immunogenic protein/peptide described in claim 3, the polynucleotide described in claim 4 and the immunogenic composition described in claim 5 or 6 are used in the preparation of prevention or treatment of SARS-CoV-2 mutant strains caused by Use in vaccines or medicines for diseases, preferably, the mutant strain is a high-risk mutant strain;

Preferably, mutant strains are containing L18F, T19i, T19R, L24DEL, P25DEL, P26DEL, A27S, A67V, H68DEL, H69DEL, V70DEL, D80A, T95i, G142D, V144DEL, Y145DEL, E156G, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15, F15 7DEL, R158DEL, N211DEL, L212I, V213G, ins214EPE, D215G, L242del, A243del, L244del, R246I, G339D, R346K, S371F, S371L, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, L 452R, S477N, T478K, E484A, E484K, E484Q, F486V, Q493R, G496S, Q498R, N501Y, Y505H, T547K, A570D, D614G, H655Y, N679K, P681H, P681R, A701V, T716I, N764K, D796Y, N856K, D950N, Q9 54H, N969K, L981F, S982A and D1118H High-risk mutant strains with at least any mutation in;

Preferably, the strain is selected from D614G mutant strain, Alpha strain, Beta strain, Delta strain, Gamma strain, Epsilon strain, BA.1 strain, BA.1.1 strain, BA.2 strain, At least one of BA.2.12.1 strain, BA.3 strain and/or BA.4/5 strain;

More preferably, the strain is selected from Alpha strain, Beta strain, Delta strain, BA.1 strain, BA.1.1 strain, BA.2 strain, BA.2.12.1 strain, BA.3 strain and/or at least one of the BA.4/5 strains.