CN118119646A

CN118119646A - Preparation and application of recombinant five-component novel coronavirus trimer protein vaccine capable of inducing broad-spectrum neutralization activity

Info

Publication number: CN118119646A
Application number: CN202380013962.8A
Authority: CN
Inventors: 谢良志; 孙春昀; 张延静
Original assignee: Sinocelltech Ltd
Current assignee: Sinocelltech Ltd
Priority date: 2022-08-08
Filing date: 2023-08-03
Publication date: 2024-05-31
Also published as: WO2024032468A1

Abstract

The invention relates to the field of molecular vaccinology, and provides a recombinant multicomponent novel coronavirus trimer protein vaccine capable of inducing broad-spectrum neutralization activity. Recombinant protein components include, but are not limited to, alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and the extracellular domain (ECD) of Omacron (BA.1, BA.4/BA.5) spike protein (S protein) homotrimeric proteins formed by introducing mutation sites and trimerization assistance structures. The multicomponent vaccine comprises ECD trimeric proteins of the above variants, either alone or in any combination, and pharmaceutically acceptable adjuvants. The vaccine combination shows excellent immunogenicity in mice and can maintain humoral immunity and cellular immune response for a long time. The multicomponent novel coronavirus trimeric protein vaccine can be used for preventing infection-related diseases caused by infection of novel coronaviruses and variants thereof.

Description

Preparation and application of recombinant five-component novel coronavirus trimer protein vaccine capable of inducing broad-spectrum neutralization activity

Cross Reference to Related Applications

The present application claims the benefit of chinese patent application 202210946805.X filed on 08, 2022, the contents of which are incorporated herein by reference.

Technical Field

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

Background

The novel coronavirus (SARS-CoV-2) has strong transmission capability, and the safe and effective vaccine is the most powerful technical means for controlling epidemic situation. Vaccines can be classified into the following categories, depending on the target and technology: inactivated vaccines, recombinant protein vaccines, viral vector vaccines, RNA vaccines, live attenuated vaccines, and virus-like particle vaccines. Since the pandemic of SARS-CoV-2, new crown vaccines developed in various countries have reached more than 200. By day 03 of 8 of 2022, 40 vaccines have been approved or conditionally used worldwide, and another 210 vaccines have entered the clinical study (https:// covid19.Trackvaccines org/vaccines /).

SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin converting enzyme 2 (ACE 2) ^[1]. The trimeric Spike protein (Spike) of the virus, after binding to the ACE2 receptor, is cleaved by host proteases into an S1 polypeptide comprising a receptor binding domain (Receptor binding domain, RBD) and an S2 polypeptide ^[2] responsible for mediating fusion of the virus with the cell membrane. The S protein is the main component of the viral envelope and plays an important role in receptor binding, fusion, viral entry and host immune defenses. The RBD region of the S protein contains a major neutralizing antibody epitope that stimulates B cells to produce high titer neutralizing antibodies against RBD. In addition, the S protein also contains abundant T cell epitopes, and can induce T cells to generate specific CTL responses and clear virus infected cells. Thus, the S protein is the most critical antigen for new coronal vaccine design. Most vaccines currently designed select either the S protein or the RBD domain protein as the core immunogen.

SARS-CoV-2 is an RNA single-stranded virus that is subject to deletion mutations and such mutations occur in multiple repeats of the S protein (Recurrent deletion regions, RDRs). Deletions or mutations may alter the conformation of the S protein such that antibodies induced by previous vaccine immunization reduce binding and neutralization of the mutant S protein resulting in reduced vaccine immunization effects and viral immune escape. Early D614G mutations (b.1) enhanced the affinity of the S protein for ACE2 receptor and became rapidly prevalent strains, but the mutations did not decrease the sensitivity ^[ ³ ^, ⁴ ^] to neutralizing antibodies. However, with the pandemic of SARS-CoV-2, 5 variants of high concern (Variants of Concern, VOC) appear worldwide: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and Omacron (B.1.1.529) and 2 careless variants (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 (increase hospitalization or mortality), severely reduce antibody neutralization by past infections or immunizations, reduce therapeutic or vaccine efficacy, or disable diagnostic assays ^[ ⁵ ^]. Alpha spreads rapidly and increases the 61% associated risk of death ^[ ⁶ ^]. The neutralization effect research results show that the neutralization capacity of the blood plasma of a convalescent person or the blood serum of a vaccine immunity person on Alpha is basically unchanged, but the neutralization capacity on Beta is greatly reduced ^[ ^7-12 ^]. Clinical results also show that Alpha has little effect on the protective effect of the vaccine, while Beta greatly reduces the protective effect ^[ ^13-16 ^] on mild symptoms. Compared with original virus and early variant strain, the Delta variant has stronger transmission capacity, short latency period and quick disease development process, and can reduce the protection effect of the vaccine.

The omacron lineage of SARS-CoV-2 has evolved multiple subtypes. Ba.1 predominates in the initial infection but is soon replaced worldwide by ba.2. Two new omacron lineages were reported, named ba.4 and ba.5, respectively, in the last 4th 2022. Ba.4 and ba.5 contain the same S sequence, although closely related to ba.2, they contain more mutations ^[17] in their RBD domains. The greater ability of ba.4/ba.5 to escape and neutralize antibodies suggests that ba.4/ba.5 is more likely to spread ^[17-19] than either ba.1 or ba.2 among vaccinators or those who break through the infection. BA.4/BA.5 has now become the dominant epidemic strain worldwide. The current vaccines are all designed based on the sequence of the early epidemic strain (its genomic sequence: genBank Accession No. NC-045512), and in view of the high transmissibility of the Alpha, delta, omicron novel subtype and the adverse effects of Beta and Omicron on the protective efficacy of existing vaccines, there is a strong need for new multicomponent vaccines with high protective efficacy against high risk variants.

Disclosure of Invention

Based on the above need for a vaccine with high protective effect against the novel coronavirus SARS-CoV-2 variant,

In a first aspect, the invention provides a method for improving the immunogenicity/antigen trimer stability of an ECD antigen of a SARS-CoV-2 mutant strain by constructing an ECD antigen comprising the amino acid sequence shown as SEQ ID No. 8, or an immunogenic fragment and/or immunogenic variant thereof, thereby

ECD is in the form of a trimer in a stable prefusion conformation;

preferably, the mutant strain is a high risk mutant strain comprising at least any one of A67V,Δ69-70,T95I,G142D/Δ143-145,Δ211/L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K、H655Y、H679Y、N764K、D796V、N856K、Q954H、N969H、L981F、T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H,N969K;

in one embodiment, the strain is Omicron (BA.4/BA.5);

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

Aluminum adjuvants, oil emulsion adjuvants, toll-like receptor (TLR) agonists, combinations of immunopotentiators, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposomal adjuvants, chinese herbal adjuvants, and small peptide adjuvants;

in one embodiment, the oil emulsion adjuvant comprises a squalene component;

Toll-like receptor (TLR) agonists comprise CpG or monophosphoryl lipid a (MPL) adsorbed on aluminium salts); and

The combination of immunopotentiators comprises QS-21 and/or MPL.

In another aspect, the invention provides a method for improving the stability of the ECD antigen immunogenicity// antigen trimer of a SARS-CoV-2 mutant strain by

Constructing a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID No. 8, or an immunogenic fragment and/or immunogenic variant thereof,

Thereby expressing a stable prefusion conformational trimer form ECD;

In one embodiment, the mutant strain is a high risk mutant strain comprising at least any one of A67V,Δ69-70,T95I,G142D/Δ143-145,Δ211/L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K、H655Y、H679Y、N764K、D796V、N856K、Q954H、N969H、L981F、T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H and N969K;

In one embodiment, the strain is Omicron (BA.4/BA.5),

In one embodiment, a polynucleotide comprising the nucleotide sequence shown as SEQ ID No. 7 or a fragment thereof is constructed.

In another aspect, the invention provides an SARS-CoV-2 mutant strain ECD immunogenic protein/peptide having improved stability of immunogenicity/antigen trimer, characterized in that the immunogenic protein/peptide comprises the amino acid sequence shown as SEQ ID No. 8, or an immunogenic fragment and/or immunogenic variant thereof,

The ECD immunogenic protein/peptide is in the form of a trimer in a stable prefusion conformation;

In one embodiment, the strain is Omicron (BA.4/BA.5).

In another aspect the invention provides a polynucleotide encoding an immunogenic protein/peptide as hereinbefore described,

In one embodiment, the nucleotide sequence shown as SEQ ID No. 7 is included.

In another aspect, the invention provides an immunogenic composition comprising

Immunogenic proteins/peptides as described previously, or

Polynucleotides as described hereinbefore, and

Any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent;

Optionally, an adjuvant is included.

Another aspect of the present invention provides a multivalent immunogenic composition characterized by further comprising

At least any one of the amino acid sequences shown in SEQ ID No. 16, SEQ ID No. 20, SEQ ID No. 28 or SEQ ID No. 32, or an immunogenic fragment and/or immunogenic variant thereof, or

At least one of the nucleotide sequences shown as SEQ ID No. 15, SEQ ID No. 19, SEQ ID No. 27 or SEQ ID No. 31 or an active variant thereof.

In one embodiment, the adjuvant of the immunogenic composition is selected from one or more of the following:

in one embodiment, the oil emulsion adjuvant comprises a squalene component;

The combination of immunopotentiators comprises QS-21 and/or MPL.

Another aspect of the present invention provides the use of the aforementioned immunogenic proteins/peptides, polynucleotides and immune complexes to prevent or treat diseases caused by SARS-CoV-2 mutant strains, preferably the mutant strains are high risk mutant strains;

in one embodiment, the mutant strain is a high risk mutant strain comprising at least any one of A67V,Δ69-70,T95I,G142D/Δ143-145,Δ211/L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K、H655Y、H679Y、N764K、D796V、N856K、Q954H、N969H、L981F、K417N、L452R、E484K、E484Q、N501Y、T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H and N969K;

In one embodiment, the strain comprises the D614G mutation (B.1), beta (B.1.351), alpha (B.1.1.7), delta (B.1.617.2), P.1, B.1.427, B.1.429 and/or Omicron (BA.1, BA.4/BA.5);

In one embodiment, the strain comprises Alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and Omicron (BA.1, BA.4/BA.5).

In another aspect, the invention provides the use of the foregoing immunogenic protein/peptide polynucleotides and immune complexes in the manufacture of a vaccine or medicament for the prevention or treatment of a disease caused by a SARS-CoV-2 mutant strain, in one embodiment the mutant strain is a high risk mutant strain;

In one embodiment, the strain comprises the D614G mutation (B.1), beta (B.1.351), alpha (B.1.1.7), delta (B.1.617.2) P.1, B.1.427, B.1.429 and/or Omicron (BA.1, BA.4/BA.5);

In one embodiment, the strain comprises Alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and/or Omicron (BA.1, BA.4/BA.5).

In another aspect, the invention provides an engineered SARS-CoV-2BA.4/BA.5ECD sequence comprising the amino acid sequence depicted by SEQ ID No. 6 or an immunogenic fragment and/or immunogenic variant thereof.

In a further aspect the present invention provides a polypeptide encoding an immunogenic protein/peptide amino acid sequence as described above or an immunogenic fragment and/or immunogenic variant thereof, preferably a nucleotide sequence as set out in SEQ ID No. 5 or an immunogenic fragment and/or immunogenic variant thereof.

Drawings

FIG. 1 is a schematic representation of the primary structure (A) and the higher structure (B, reference PDB:6 XLR) of the engineered S-ECD.

FIG. 2 is a diagram showing the mutation sites of Omicron (BA.4/BA.5) variant.

FIG. 3 shows S-Trimer-TM41C protein purity analysis; (a) a non-reducing SDS-PAGE representative profile; (B) SEC-HPLC representative profile.

FIG. 4 shows serum antibody titer assays (GeoMean+ -SD) after immunization of C57BL/6 mice with TM41 and TM41C single component vaccines.

FIG. 5 shows serum antibody titer assays (GeoMean+ -SD) after immunization of C57BL/6 mice with SCTV01E and SCTV01E-1 vaccines.

Detailed Description

Definition of the definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as 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," "the," and "the" include plural referents unless the context clearly dictates otherwise.

The terms "comprising," "including," and "containing" are intended to include the specific components without excluding any other components. Such as "consisting essentially of … …" allows for the inclusion of other components or steps that do not detract from the novel or essential features of the invention, i.e., they exclude other non-recited components or steps that do. The term "consisting of … …" is meant to include a particular ingredient or component and exclude all other ingredients.

The term "antigen" refers to a foreign substance recognized (specifically bound) by an antibody or T cell receptor, but which does not definitively induce an immune response. Foreign substances that induce specific immunity are called "immunogenic antigens" or "immunogens". "hapten" refers to an antigen that is not itself capable of eliciting an immune response (although a conjugate of several molecular haptens, or a conjugate 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 generation of antibodies that recognize and bind with some affinity to antigens in the immunogenic compositions of the invention, a "cell-mediated immune response" being an immune response mediated by T cells and/or other leukocytes. A "cell-mediated immune response" is induced by providing an epitope associated with a class I or class II molecule of the Major Histocompatibility Complex (MHC), CD1 or other atypical MHC-like molecule.

The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, such as a microorganism or component thereof, which composition can be used to elicit an immune response in an individual.

"Immunogenicity" as used herein means the ability of an antigen (or epitope of an antigen), such as a coronavirus spinous process protein receptor binding region or an immunogenic composition, to elicit a humoral or cell-mediated immune response or both in a host (e.g., a mammal).

"Protective" immune response refers to the ability of an immunogenic composition to elicit a humoral or cell-mediated immune response, or both, for protecting an individual from infection. The protection provided need not be absolute, i.e., it need not completely prevent or eradicate the infection, so long as there is a statistically significant improvement over a control population of individuals (e.g., infected animals that are not administered a vaccine or immunogenic composition). Protection may be limited to alleviating the severity of symptoms of infection or rapidity of onset.

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

The effectiveness of an antigen as an immunogen may be measured, for example, by a proliferation assay, by a cytolytic 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 consecutive 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 a modified backbone. It is to be understood that the present invention provides polynucleotides comprising sequences complementary to the sequences described herein. "Polynucleotide" as contemplated in the present invention includes forward strands (5 'to 3') and reverse complementary strands (3 'to 5'). Polynucleotides according to the invention may be prepared in different ways (e.g., by chemical synthesis, by gene cloning, etc.), and may take various forms (e.g., linear or branched, single-or double-stranded, or hybrids thereof, primers, probes, etc.).

The term "immunogenic protein/peptide" includes polypeptides having immunological activity in the sense that upon administration to a host they are capable of eliciting an immune response against the humoral and/or cellular type of the protein. Thus, a protein fragment according to the invention comprises or essentially consists of at least one epitope or antigenic determinant. As used herein, an "immunogenic" protein or polypeptide includes the full-length sequence of a protein, an analog thereof, or an immunogenic fragment thereof. An "immunogenic fragment" refers to a fragment of a protein that contains one or more epitopes, thereby eliciting an immune response as described above.

The term "immunogenic protein/peptide" also encompasses deletions, additions and substitutions to the sequence, provided that the polypeptide functions to produce an immune response as defined herein, i.e. "immunogenic variants".

The term "active variant of a nucleotide sequence" also encompasses deletions, additions and substitutions to the sequence, provided that the nucleotide sequence functions to produce an immune response as defined herein.

The SCTV01E-1 recombinant protein vaccine provided by the invention is modified based on an extracellular domain (ECD, containing S1 and S2 parts) of SARS-CoV-2 spike protein. The known natural spike protein of SARS-CoV-2 is in a trimeric structure, and in the process of generating and performing infection function, the membrane fusion process is completed by the protease in the Golgi apparatus and on the cell surface being easily cut off by the RRAR site existing between S1 and S2, and then S1 is shed, and further the S2 structure is converted from prefusion conformation to postfusion conformation, so that the membrane fusion ^[ ²⁰ ^] is completed. Based on this, ECD trimer with prefusion stable conformation is the next step, and in order to obtain ECD trimer with stable prefusion conformation, the present invention is modified based on S protein of different strain variants in three parts as follows:

It has now been found that antibodies with higher neutralizing activity bind to both the S1 region (specifically to the NTD and RBD regions in S1). The S1 part is kept intact, and is important for the generation of neutralizing antibodies induced by the novel crown vaccine. The Furin locus is removed by modification in the SCTV01E-1 recombinant protein vaccine, namely, the amino acid sequence from 679 to 688 is fixed to NSPGSASSVA, so that the possibility of S1 breakage and shedding is reduced.

2) The prefusion conformation of spike protein is unstable due to the allosteric propensity of S2 itself, whereas effective induction of neutralizing antibodies requires maintenance of prefusion conformation stable, which has been demonstrated ^[ ²¹ ^, ²² ^] in RSV and HIV-1 vaccine studies. In current marketed vaccines, the modification ^[ ^23-25 ^] of S-2P (i.e., mutation of the 986 and 987 amino acids to proline) is commonly employed. In addition, the invention also introduces HexaPro mutation (namely mutation of 817, 892, 899 and 942 amino acids into proline in addition to S-2P mutation) ^[ ²⁶ ^] which can effectively improve stability and does not influence the three-dimensional structure of the mutant. These mutation sites are all located at the N-terminal or Loop region of the alpha-helix in S2, and after mutation into the proline (P) type with this secondary structure propensity, the allosteric propensity of S2 can be effectively reduced to stabilize the prefusion conformation of S2.

3) Finally, in order to further stabilize the S-ECD trimer structure, the invention adds a trimerization module T4foldon at the C-terminal of the vaccine molecule. This module is derived from the C-terminal domain of the fibrin of the T4 bacteriophage, with 27 amino acids.

After the recombinant S-ECD trimeric protein antigen modified as described above is recombined into an expression vector and the expressed recombinant S-ECD trimeric protein is subjected to conventional purity and stability analysis, corresponding trimeric proteins, namely D614G epidemic TM8 protein, alpha variant TM22 protein, beta variant TM23 protein, delta variant TM28 protein, omicron BA.1 variant TM41 protein and Omicron BA.4/BA.5 variant TM41C protein, are prepared.

The prepared D614G epidemic strain TM8 protein vaccine is used for immunizing mice, and then immunological determination is carried out on Beta variant strain TM23 protein vaccine in cynomolgus monkeys, and immunological determination on Alpha variant strain TM22 protein vaccine in mice shows that the three vaccines prepared by the invention can generate antibody immune response with sufficient titer in experimental animals; and in the immunological evaluation of mice, the use of a TM8+TM23 double-component vaccine and a TM22+TM23 double-component vaccine (SCTV 01C) also indicates that the double-component vaccine has higher and similar neutralization titers on different strains, so that the double-component vaccine has better broad-spectrum neutralization capability compared with a single-component vaccine, and the neutralization titers of the double-component vaccine on different variant strains are far higher than the neutralization titers of serum of a convalescence person on early epidemic strains (the genome sequence of the double-component vaccine is GenBank Accession No. NC_ 045512). Further studies have shown that the protective efficacy of the TM22+TM23+TM28+TM41 four-component vaccine (SCTV 01E) is superior to that of the TM22+TM23 two-component vaccine when protecting against Delta and Omicron variant infections.

The SCTV01E-1 recombinant protein vaccine is modified based on the extracellular domain (ECD, containing S1 and S2 parts) of SARS-CoV-2 spike protein, and is a TM22+TM23+TM28+TM41+TM41C five-component vaccine. The natural spike protein of SARS-CoV-2 is a trimer structure, during the production and infection function, the RRAR site existing between S1 and S2 is easy to be cut by protease in Golgi body and on the surface of cell, then S1 is fallen off, further S2 structure is converted from prefusion conformation to postfusion conformation, thus completing the membrane fusion process ^[20]. In order to obtain ECD trimers with stable prefusion conformations, the present invention was modified based on S protein of different strain variants in three parts as follows (Table 1 and FIG. 1):

1) It has now been found that antibodies with higher neutralizing activity bind to both the S1 region (specifically to the NTD and RBD regions in S1). The S1 part is kept intact, and is important for the generation of neutralizing antibodies induced by the novel crown vaccine. The Furin locus is removed by modification in the SCTV01E-1 recombinant protein vaccine, namely, the amino acid sequence from 679 to 688 is fixed to NSPGSASSVA, so that the possibility of S1 breakage and shedding is reduced.

2) The prefusion conformation of spike protein is unstable due to the allosteric propensity of S2 itself, whereas effective induction of neutralizing antibodies requires maintenance of prefusion conformation stable, which has been demonstrated ^[ ²¹ ^, ²² ^] in RSV and HIV-1 vaccine studies. The majority of new coronal vaccines currently in the market or clinical stage focus on the part of the spike protein that plays an important role in viral infection, thus ensuring that it is stable in the prefusion conformation is a concern. In current marketed vaccines, the S-2P (i.e. mutation of amino acids 986 and 987 to proline) modification scheme ^[ ^23-25 ^] is commonly employed. In order to further improve the prefusion conformational stability of ECD and improve the expression quantity and the product stability of ECD in CHO recombinant cells, so that the ECD is convenient to store and transport while reducing the production cost, the invention introduces HexaPro mutation (namely mutation of 817, 892, 899 and 942 amino acid into proline in addition to S-2P mutation) ^[ ²⁶ ^] which can effectively improve the stability and does not influence the three-dimensional structure of the SCTV01E-1 recombinant protein vaccine into each antigen component. These mutation sites are all located at the N-terminal or Loop region of the alpha-helix in S2, and after mutation into the proline (P) type with this secondary structure propensity, the allosteric propensity of S2 can be effectively reduced to stabilize the prefusion conformation of S2.

3) Finally, in order to further stabilize the S-ECD trimer structure, the invention adds a trimerization module T4foldon at the C-terminal of each antigen component of the vaccine molecule. This module is derived from the C-terminal domain of the fibrin of the T4 bacteriophage, with 27 amino acids. T4foldon has been used in RSV vaccine candidates and demonstrated good safety ^[ ²⁷ ^] in clinical phase I studies.

TABLE 1 molecular structural design modification of antigenic components of SCTVE-1 vaccine

The ECD trimeric immunogenic proteins/peptides of the invention exhibit excellent immunogenicity in mice and maintain long-term humoral and cellular immune responses.

Examples

Example 1: new coronavirus recombinant spike protein extracellular region (S-ECD) trimer protein antigen design, construction of expression vector and protein production

1.1 Construction of S-ECD trimeric protein (TM 41C) expression vector based on Omicon (BA.4/BA.5) sequence (EPI_ISL_ 11542270.1)

FIG. 2 is a diagram showing the mutation sites of Omicron (BA.4/BA.5) variant. The mutation site comprises ：T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H,N969K(https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/).

TM41C contains a 3693bp gene fragment, which was obtained by PCR from the template pSE-CoV2-S-ECDTM 41C-T4F-primer. The expression vector of pGS5-9-CoV2-S-ECDTM 41C-T4F-primer is obtained by constructing the expression vector into a stable pGS5-9-A1 strain which is digested with HindIII and EcoRI by an In-fusion method.

Amplification primers

1.2 Expression and purification of TM41C trimer proteins

The target gene constructed above is transferred into HD-BIOP3 (GS-) cells (horizons) by a chemical method, and cultured by adopting an independently developed serum-free culture medium, a cell strain with stable expression is obtained by MSX pressurized screening, and after 14 days of feeding culture, a culture supernatant is 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 adopting an anion chromatography (NanoGel-50Q, nanocom) combination mode and a mixed anion chromatography (DiamondMIX-A, bognong) flow-through mode to carry out further purification, and removing impurities related to products and processes; next, viruses were inactivated and removed using low pH incubation and virus removal filtration (Planova), and finally ultrafiltration exchange with ultrafiltration membrane pack (Millipore) to citrate buffer. S-ECD trimer expression levels >500mg/L.

Example 2: analysis of purity and stability of recombinant spike protein extracellular region (S-ECD) trimer protein of novel coronavirus

2.1 Analysis of purity of recombinant TM41C trimer protein

The purified recombinant S-ECD trimer protein stock solution was placed in a buffer containing 1.7mM citric acid, 8mM sodium citrate, 300mM sodium chloride, 0.3g/kg polysorbate 80, pH 7.0.+ -. 0.2, at a concentration of about 0.79mg/mL, and analyzed for primary structure purity and trimer content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS polyacrylamide gel electrophoresis, SDS-PAGE) and by size exclusion high performance liquid chromatography (size-exclusion high performance liquid chromatograph, SEC-HPLC), and morphological characteristics by dynamic light scattering (DYNAMIC LIGHT SCATTERING, DLS).

Specific operation steps of SDS-PAGE: (1) preparation of SDS-PAGE gel: 3.9% of concentrated gum and 7.5% of separation gum; (2) boiling the sample at 100 ℃ for 2min, centrifuging and loading 8 mug; (3) Coomassie brilliant blue staining followed by bleaching. The SEC-HPLC operation steps are: (1) apparatus: a liquid chromatography system (Agilent company, model: agilent 1260), a water-soluble size exclusion chromatography column (Sepax company, model: SRT-C SEC-500 chromatography column); (2) mobile phase: 200mM NaH ₂PO₄, 100mM Arginine,pH 6.5,0.01% isopropyl alcohol (IPA); (3) the loading amount is 80 mug; (3) The detection wavelength was 280nM, the analysis time was 35min and the flow rate was 0.15mL/min.

The specific operation steps of DLS are as follows: (1) apparatus: dynamic light scattering instrument (Wyatt Technology Co., model number DynaPro NanoStar); (2) a loading amount of 50. Mu.L; (3) After data collection, data was analyzed using Dynamics 7.1.8 software.

The recombinant TM41C protein is a homotrimeric structure due to its non-covalent hydrophobic interactions. The non-reducing SDS-PAGE treatment to obtain monomer molecule with molecular weight of 148kDa (figure 3) with purity of 95.2%; SEC-HPLC shows that the trimer purity is 97.2%, the aggregate and fragment proportion content is less than 5%, and the main peak molecular weight is 512kDa on average; dynamic light scattering results showed that the average radius of the TM41C trimeric protein molecule was 8.1nm (Table 2).

TABLE 2 recombinant S-ECD trimer purity analysis

2.2 Evaluation of stability of recombinant TM41C trimer protein

The recombinant TM41C trimeric protein was stored at 37℃for 1 week (37T 1W), and after storage at 80℃for 8 hours, the recombinant TM41C trimeric protein was thawed at 45℃for 0.5h (F/T), and repeated freeze thawing was performed 5 times, and the changes in the trimer content were analyzed by SDS-PAGE and SEC-HPLC, and the data are shown in Table 3.

As shown in Table 3, after the recombinant TM41C trimeric protein is accelerated for 1 week at 37 ℃ and after 5 times of repeated freeze thawing, the purity of non-reduced SDS-PAGE and the content of SEC-HPLC trimer are both above 88.0%, the purity change after acceleration is within 2.0%, the aggregate and the fragment are not obviously increased, and good heat acceleration stability and freeze thawing stability are shown.

TABLE 3 evaluation of recombinant TM41C trimer protein stability

Example 3: TM41 single-component vaccine and multicomponent vaccineImmunological evaluation of mice

3.1 Vaccine preparation and immunization protocol

Expression and purification of the TM22 and TM23 trimeric proteins are described in the patent "a method for improving the immunogenicity of the ECD antigen/stability of the antigen trimer of the SARS-CoV-2 mutant strain" (application No.: PCT/CN2022/095609 and its priority application Nos.: 202110606512.2 and 202111237604.4, incorporated herein in their entirety). Applicants have elaborated in this patent that the two-component vaccine composed of TM22+ TM23 has a more excellent broad spectrum neutralization capacity than the TM22 and TM23 single-component vaccines. Expression and purification of TM28 trimeric proteins see patent application No. PCT/CN2022/107213 and its priority application No. 202110838359.6, incorporated herein in its entirety, for the preparation and use of a recombinant multicomponent novel coronavirus trimeric protein vaccine capable of inducing broad-spectrum neutralization activity. Applicant elaborates in this patent application that trivalent vaccine compositions of tm22+tm23+tm28 have a more excellent broad spectrum neutralization capacity than single component vaccines. Expression and purification of TM41 trimeric proteins see patent application "preparation and use of a recombinant multicomponent novel coronavirus trimeric protein vaccine capable of inducing broad-spectrum neutralization activity" (application number: PCT/CN2023/078135 and its priority application number: 202210184528.3, incorporated herein in its entirety). Applicant elaborates in this patent application that tetravalent seedlings of the composition tm22+tm23+tm28+tm41 have a more excellent broad spectrum neutralization capacity than single component vaccines. To further broaden the broad spectrum neutralization effect of the vaccine, especially against the Omicron new subtype variant, the applicant added TM41C component on the basis of TM22+ TM23+ TM28+ TM41 tetravalent vaccine to make up a five-component vaccine.

Single or multicomponent vaccine samples were prepared by pre-diluting purified TM22, TM23, TM28, TM41 and TM41C trimeric proteins with PBS according to the final immunization dose (table 4) and mixing with MF59 (8×, source: shenzhou cell engineering ltd, supra) in equal volumes.

Table 4 summary of immune packet information

3.2 Immunization of mice

Female C57BL/6 mice (source: beijing Vitre Liwa laboratory animal technologies Co., ltd., body weight 18-20 g) from 6-8 weeks were intramuscular injected with 0.1mL of vaccine sample containing MF59 adjuvant. A total of 2 immunizations were performed with an immunization interval of 14 days. The orbit was collected for 14 days after the first immunization (14 days 1 and 7 days after 2 immunization (7 days 2) and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immunoassay.

3.3 Determination of neutralizing titers in mouse immune serum

The 7 day immune serum of different dilution was added at 50. Mu.L/well to 96-well plate, then 100-200 TCID ₅₀ Omicron (BA.1), omicron (BA.2.12.1), omicron (BA.2.75) or Omicron (BA.4/5) pseudovirus (pseudovirus was prepared by amplifying in cell line expressing Spike and mutant protein thereof using replication defective vesicular stomatitis virus (i.e., VSV.DELTA.G-Luc-G) in which VSV-G protein gene was replaced with luciferase reporter gene in viral genome as vector, prepared by Shenzhou cell engineering Co., ltd., hereinafter, and incubated in a 5% CO ₂ incubator at 37℃for 1h after mixing. The wells without serum to which the pseudoviruses were added were used as positive controls, and the wells without serum and pseudoviruses were used as negative controls. After the incubation, 100. Mu.L/well of 2X 10 ⁴ Huh-7 cells were inoculated, mixed and placed in a 5% CO ₂ incubator at 37℃for stationary culture for about 20 hours. After the completion of the culture, the culture supernatant was removed, and 1X PASSIVE LYSIS buffer was added to 50. Mu.L/well, followed by mixing and cell lysis. The neutralization rate was calculated by taking 40. Mu.L/Kong Zhuairu of a 96-well all-albino chemiluminescent plate, adding a luciferase substrate to 40. Mu.L/well using an LB960 microplate type luminometer and measuring the luminescence value (RLU). Neutralization rate = (positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs) ×100%, IC ₅₀ was calculated according to Reed-Muench formula, i.e. neutralization potency NAT ₅₀.

As shown in FIG. 4, the results of the serum neutralizing antibody test for 2-day immunization against 7 days show that the neutralizing antibody titres (geometric mean value NAT ₅₀) induced by TM41 single component vaccine against Omicron BA.1, BA.2, BA.2.12.1, BA.2.75 and BA.4/5 were 17277, 2520, 873, 72 and 100, respectively, the NAT ₅₀ induced by TM41 single component vaccine against the above variant strains was 108, 4319, 7678, 1433 and 33518, respectively, and the NAT ₅₀ induced by TM41 single component vaccine against Omicron BA.1, BA.2, BA.2.12.1, BA.2.75 and BA.4/5 was 1/159.9 times, 1.7 times, 8.7 times, 14.3 times and 493.4 times, respectively. Demonstrating that the TM41C single component vaccine has a broader neutralizing activity against the Omacron novel subtype variant than the TM41 single component vaccine.

NAT ₅₀ for omicon ba.1, ba.2.12.1, ba.2.75 and ba.4/5 for SCTV01E vaccine induction was 4633, 543, 207 and 123 respectively, NAT ₅₀ for the above variant strains for SCTV01E vaccine induction was 1243, 6789, 2498 and 16194 respectively, NAT ₅₀ for omicon ba.1, ba.2.12.1, ba.2.75 and ba.4/5 for SCTV01E vaccine induction was 0.3 times, 12.5 times, 12.0 times and 131.6 times respectively (fig. 5). It was demonstrated that the SCTV01E-1 vaccine has a broader spectrum of neutralizing activity against the different variants of SARS-CoV-2Omicron BA.2.12.1, BA.2.75 and BA.4/5 than the SCTV01E vaccine. Among them, FIG. 5 shows the results of serum antibody titer detection (GeoMean.+ -. SD) after immunization of C57BL/6 mice with SCTV01E and SCTV01E-1 vaccines.

In conclusion, the five-component vaccine has broad-spectrum neutralization capability against Omicron new subtype variants, and is hopeful to generate cross protection capability to various variant strains due to covering more variant sites, so that the protection rate to variant infection is improved.

While the invention has been described in detail in the foregoing description and with reference to the embodiments, it is for an understanding of the invention to be readily apparent to those skilled in the art that various modifications and improvements can be made to the technical solution of the invention without departing from the spirit or scope of the appended claims.

Sequence list

Reference to the literature

1.Zhou,P.,et al.,Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin.BioRxiv,2020.

2.Hoffmann,M.,et al.,SARS-CoV-2cell entry depends on ACE2and TMPRSS2and is blocked by a clinically proven protease inhibitor.Cell,2020.

3.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.

4.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.

5.https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html.

6.Davies,N.G.,et al.,Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7.Nature,2021.

7.Cele,S.,et al.,Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma.2021.

8.Zhou,D.,et al.,Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.Cell,2021.

9.Tada,T.,Neutralization of viruses with SARS-CoV-2 variant spike proteins by convalescent sera and BNT162b2 mRNA vaccine-elicited antibodies.2021.

10.Wang,P.,et al.,Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization.bioRxiv,2021.

11.Wadman,M.and J.Cohen,Novavax vaccine delivers 89％efficacy against COVID-19 in UK—but is less potent.Science,2021.

12.Wu,K.,et al.,mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants.2021.

13.Madhi,S.A.,et al.,Efficacy of the ChAdOx1 nCoV-19 Covid-19 Vaccine against the B.1.351 Variant.2021.

14.Shinde,V.,et al.,Efficacy of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 Variant.New England Journal of Medicine,2021.

15.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.

16.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.

17.Tuekprakhon,A.,et al.,Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum.Cell,2022.185(14):p.2422-2433.e13.

18.Cao,Y.,et al.,BA.2.12.1,BA.4 and BA.5 escape antibodies elicited by Omicron infection.Nature,2022:p.1-3.

19.Arora,P.,et al.,Augmented neutralisation resistance of emerging omicron subvariants BA.2.12.1,BA.4,and BA.5.The Lancet Infectious Diseases,2022.22(8):p.1117-1118.

20.Cai,Y.,J.Zhang,and T.Xiao,Distinct conformational states of SARS-CoV-2 spike protein.2020.369(6511):p.1586-1592.

21.McLellan,J.S.,et al.,Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus.Science,2013.342(6158):p.592-8.

22.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.

23.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.

24.Mercado,N.B.,et al.,Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques.2020.586(7830):p.583-588.

25.Corbett,K.S.,et al.,SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness.bioRxiv,2020.

26.Hsieh,C.L.,et al.,Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes.bioRxiv,2020.

27.Crank,M.C.,Aproof of concept for structure-based vaccine design targeting RSV in humans.2019.365(6452):p.505-509.

Claims

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

Construction of ECD antigen comprising the amino acid sequence shown as SEQ ID No. 8, or an immunogenic fragment and/or immunogenic variant thereof, thereby

ECD is in the form of a trimer in a stable prefusion conformation;

Preferably, the mutant strain is a high risk mutant strain comprising at least any one of A67V,Δ69-70,T95I,G142D/Δ143-145,Δ211/L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K、H655Y、H679Y、N764K、D796V、N856K、Q954H、N969H and L981F、T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H,N969K;

preferably, the strain is Omicron (BA.4/BA.5);

Preferably, the ECD antigen is co-administered to the subject with one or more adjuvants selected from the group consisting of:

Aluminum adjuvants, oil emulsion adjuvants, toll-like receptor (TLR) agonists, combinations of immunopotentiators, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposomal adjuvants, chinese herbal adjuvants, and small peptide adjuvants;

Preferably, the oil emulsion adjuvant comprises a squalene component;

Toll-like receptor (TLR) agonists comprise CpG or monophosphoryl lipid a (MPL) adsorbed on an aluminium salt; and

The combination of immunopotentiators comprises QS-21 and/or MPL.
A method of increasing the immunogenicity// antigen trimer stability of a SARS-CoV-2 mutant strain ECD antigen by constructing a polynucleotide encoding an amino acid sequence set forth in SEQ ID No. 8, or an immunogenic fragment and/or immunogenic variant thereof, to express a stable prefusion conformational form of the ECD;

Preferably, the mutant strain is a high risk mutant strain comprising at least any one of A67V,Δ69-70,T95I,G142D/Δ143-145,Δ211/L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K、H655Y、H679Y、N764K、D796V、N856K、Q954H、N969H、L981F、T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H and N969K;

more preferably, the strain is omacron (ba.4/ba.5);

Most preferably, a polynucleotide comprising the nucleotide sequence shown as SEQ ID No. 7 or a fragment thereof is constructed.
An immunogenic protein/peptide of SARS-CoV-2 mutant strain ECD with improved stability of immunogenicity/antigen trimer, characterized in that the immunogenic protein/peptide comprises the amino acid sequence shown in SEQ ID No. 8, or an immunogenic fragment and/or immunogenic variant thereof,

The ECD immunogenic protein/peptide is in the form of a trimer in a stable prefusion conformation;

Preferably, the mutant strain is a high risk mutant strain comprising at least any one of A67V,Δ69-70,T95I,G142D/Δ143-145,Δ211/L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K、H655Y、H679Y、N764K、D796V、N856K、Q954H、N969H、L981F、T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H and N969K;

preferably, the strain is Omicron (BA.4/BA.5).
A polynucleotide encoding the immunogenic protein/peptide according to claim 3,

Preferably, the nucleotide sequence shown as SEQ ID No. 7 is included.
An immunogenic composition comprising

An immunogenic protein/peptide according to claim 3, or

The polynucleotide according to claim 4, and

Any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent;

Optionally, an adjuvant is included.
The immunogenic composition of claim 5, further comprising

At least any one of the amino acid sequences shown in SEQ ID No. 16, SEQ ID No. 20, SEQ ID No. 28 or SEQ ID No. 32, or an immunogenic fragment and/or immunogenic variant thereof, or

At least one of the nucleotide sequences shown as SEQ ID No. 15, SEQ ID No. 19, SEQ ID No. 27 or SEQ ID No. 31 or an active variant thereof.
The immunogenic composition of claim 5, wherein the adjuvant is selected from one or more of the following:

Aluminum adjuvants, oil emulsion adjuvants, toll-like receptor (TLR) agonists, combinations of immunopotentiators, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposomal adjuvants, chinese herbal adjuvants, and small peptide adjuvants;

Preferably, the oil emulsion adjuvant comprises a squalene component;

Toll-like receptor (TLR) agonists comprise CpG or monophosphoryl lipid a (MPL) adsorbed on an aluminium salt; and

The combination of immunopotentiators comprises QS-21 and/or MPL.
Use of the immunogenic protein/peptide of claim 3, the polynucleotide of claim 4 and the immunocomplex of claim 5 or 6 to prevent or treat diseases caused by SARS-CoV-2 mutant strains, preferably the mutant strains are high risk mutant strains;

Preferably, the mutant strain is a high risk mutant strain comprising at least any one of A67V,Δ69-70,T95I,G142D/Δ143-145,Δ211/L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K、H655Y、H679Y、N764K、D796V、N856K、Q954H、N969H、L981F、K417N、L452R、E484K、E484Q、 and N501Y、T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H and N969K;

Preferably, the strain comprises the D614G mutation (B.1), beta (B.1.351), alpha (B.1.1.7), delta (B.1.617.2), P.1, B.1.427, B.1.429 and/or Omicron (BA.1, BA.4/BA.5);

More preferably, the strain comprises Alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and Omacron (BA.1, BA.4/BA.5).
Use of the immunogenic protein/peptide of claim 3, the polynucleotide of claim 4 and the immunocomplex of claim 5 or 6 for the preparation of a vaccine or medicament for the prevention or treatment of a disease caused by a SARS-CoV-2 mutant strain, preferably the mutant strain is a high risk mutant strain;

Preferably, the mutant strain is a high risk mutant strain comprising at least any one of A67V,Δ69-70,T95I,G142D/Δ143-145,Δ211/L212I,ins214EPE,G339D,S371L,S373P,S375F,K417N,N440K,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y,Y505H,T547K、H655Y、H679Y、N764K、D796V、N856K、Q954H、N969H、L981F、K417N、L452R、E484K、E484Q、 and N501Y、T19I,L24del,P25del,P26del,A27S,H68del,V69del,G142D,V213G,G339D,S371F,S373P,S375F,T376A,D405N,R408S,K417N,N440K,L452R,S477N,T478K,E484A,F486V,Q498R,N501Y,Y505H,D614G,H655Y,N679K,P681H,N764K,D796Y,Q954H and N969K;

Preferably, the strain comprises the D614G mutation (B.1), beta (B.1.351), alpha (B.1.1.7), delta (B.1.617.2) P.1, B.1.427, B.1.429 and/or Omicron (BA.1, BA.4/BA.5);

More preferably, the strain comprises Alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and/or Omicron (BA.1, BA.4/BA.5).
An engineered SARS-CoV-2 BA.4/BA.5 ECD sequence comprising

The amino acid sequence shown in SEQ ID No. 6 or an immunogenic fragment and/or an immunogenic variant thereof.
An amino acid sequence encoding claim 10 or an immunogenic fragment and/or immunogenic variant thereof, preferably a nucleotide sequence as set forth in SEQ ID No. 5 or an immunogenic fragment and/or immunogenic variant thereof.