WO2024032468A1 - 一种可诱导广谱中和活性重组五组分新冠病毒三聚体蛋白疫苗的制备及应用 - Google Patents

一种可诱导广谱中和活性重组五组分新冠病毒三聚体蛋白疫苗的制备及应用 Download PDF

Info

Publication number
WO2024032468A1
WO2024032468A1 PCT/CN2023/111051 CN2023111051W WO2024032468A1 WO 2024032468 A1 WO2024032468 A1 WO 2024032468A1 CN 2023111051 W CN2023111051 W CN 2023111051W WO 2024032468 A1 WO2024032468 A1 WO 2024032468A1
Authority
WO
WIPO (PCT)
Prior art keywords
immunogenic
adjuvant
protein
seq
cov
Prior art date
Application number
PCT/CN2023/111051
Other languages
English (en)
French (fr)
Inventor
谢良志
孙春昀
张延静
Original Assignee
神州细胞工程有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 神州细胞工程有限公司 filed Critical 神州细胞工程有限公司
Publication of WO2024032468A1 publication Critical patent/WO2024032468A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention relates to the field of molecular vaccinology, and relates to the preparation and application of a recombinant multi-component novel coronavirus trimer protein vaccine that can induce broad-spectrum neutralizing activity.
  • the new coronavirus (SARS-CoV-2) has strong transmission ability, and a safe and effective vaccine is the most powerful technical means to control the epidemic.
  • 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, etc. Since the SARS-CoV-2 pandemic, more than 200 COVID-19 vaccines have been developed by various countries. As of August 3, 2022, 40 vaccines around the world have been approved for use or used with conditions, and an additional 210 vaccines have entered clinical research (https://covid19.trackvaccines.org/vaccines/).
  • SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin-converting enzyme 2 (ACE2) [1] .
  • ACE2 angiotensin-converting enzyme 2
  • the trimeric spike protein of the virus (Spike) 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 S protein contains the main neutralizing antibody epitope, which can stimulate B cells to produce high-titer neutralizing antibodies against RBD.
  • S protein also contains abundant T cell epitopes, which can induce T cells to produce specific CTL responses and clear virus-infected cells. Therefore, S protein is the most critical antigen in the design of the new coronavirus vaccine. The vast majority of vaccines currently designed use S protein or RBD domain protein as the core immunogen.
  • SARS-CoV-2 is an RNA single-stranded virus that is prone to deletion mutations, and these mutations mostly occur in the Recurrent deletion regions (RDRs) of the S protein. Deletions or mutations may change the conformation of the S protein, causing the antibodies induced by previous vaccine immunity to reduce the binding and neutralization of the mutated S protein, leading to a decrease in vaccine immunity and immune evasion of the virus.
  • the early D614G mutation (B.1) enhanced the affinity of the S protein to the ACE2 receptor and quickly became an epidemic strain, but this mutation did not reduce the sensitivity to neutralizing antibodies [ 3 , 4 ] .
  • VOC high concern
  • Beta B.1.351
  • Gamma P. 1
  • Delta B.1.617.2
  • Omicron B.1.1.529
  • VOI two variants of interest
  • Lambda C.37
  • Mu B.1.621
  • Alpha spreads rapidly and can increase the risk of related death by 61% [ 6 ] .
  • Neutralization effect study results show that the neutralizing ability of alpha by plasma of convalescent patients or serum of vaccine immune persons remains basically unchanged, but the neutralizing ability of beta is significantly reduced [ 7-12 ] .
  • Clinical results also show that Alpha has little effect on the protective effect of the vaccine, while Beta will significantly reduce the protective effect on mild disease [ 13-16 ] .
  • the Delta mutation has stronger transmissibility, shorter incubation period, faster disease progression, and can also reduce the protective effect of the vaccine.
  • BA.1 dominates initial infections but is quickly replaced globally by BA.2.
  • BA.4 and BA.5 two new Omicron lineages were reported, named BA.4 and BA.5 respectively.
  • BA.4 and BA.5 contain the same S sequence. Although closely related to BA.2, they contain more mutations in their RBD domains [17] .
  • BA.4/BA.5 has a stronger ability to escape neutralizing antibodies, indicating that BA.4/BA.5 is more likely to spread among vaccine recipients or people with breakthrough infection than BA.1 or BA.2 [17-19] .
  • BA.4/BA.5 has now become the main epidemic strain in the world.
  • the current vaccines are all designed based on the sequence of the early epidemic strain (its genome sequence: GenBank Accession No.NC_045512).
  • GenBank Accession No.NC_045512 the genome sequence of the early epidemic strain.
  • the first aspect of the present invention provides a method for improving the immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strain ECD antigen by constructing an amino acid sequence containing the amino acid sequence shown in SEQ ID No: 8, or its immune genic fragments and/or immunogenic variants of the ECD antigen, thereby
  • ECD is a trimer in a stable prefusion conformation
  • the mutant strain contains A67V, ⁇ 69-70, T95I, G142D/ ⁇ 143-145, ⁇ 211/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,
  • the strain is Omicron (BA.4/BA.5);
  • the ECD antigen is co-administered to the subject with one or more adjuvants selected from:
  • TLR Toll-like receptor
  • the oil emulsion adjuvant comprises a squalene component
  • TLR Toll-like receptor
  • MPL monophosphoryl lipid A
  • the combination of immunopotentiators includes QS-21 and/or MPL.
  • Another aspect of the present invention provides a method for improving the immunogenicity//antigen trimer stability of SARS-CoV-2 mutant strain ECD antigen, by
  • the mutant strain is A67V, ⁇ 69-70, T95I, G142D/ ⁇ 143-145, ⁇ 211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A.
  • the strain is Omicron (BA.4/BA.5),
  • a polynucleotide comprising the nucleotide sequence shown in SEQ ID No: 7 or a fragment thereof is constructed.
  • Another aspect of the present invention provides a SARS-CoV-2 mutant strain ECD immunogenic protein/peptide with improved immunogenicity/antigen trimer stability, characterized in that the immunogenic protein/peptide contains SEQ The amino acid sequence shown in ID No: 8, or its immunogenic fragment and/or immunogenic variant,
  • the ECD immunogenic protein/peptide is a trimer in a stable prefusion conformation
  • the mutant strain is A67V, ⁇ 69-70, T95I, G142D/ ⁇ 143-145, ⁇ 211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A.
  • the strain is Omicron (BA.4/BA.5).
  • Another aspect of the invention provides a polynucleotide encoding an immunogenic protein/peptide as described above,
  • nucleotide sequence shown in SEQ ID No:7 is included.
  • Another aspect of the invention provides an immunogenic composition, characterized by comprising
  • an adjuvant is included.
  • Another aspect of the invention provides a multivalent immunogenic composition, further comprising
  • the adjuvant of the immunogenic composition is selected from one or more of the following:
  • TLR Toll-like receptor
  • the oil emulsion adjuvant comprises a squalene component
  • TLR Toll-like receptor
  • MPL monophosphoryl lipid A
  • the combination of immunopotentiators includes 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 mutant strains of SARS-CoV-2.
  • the mutant strains are high-risk mutant strains. strain;
  • the mutant strain is A67V, ⁇ 69-70, T95I, G142D/ ⁇ 143-145, ⁇ 211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A.
  • the strain comprises 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);
  • the strain includes Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (BA.1, BA.4/BA.5).
  • Another aspect of the present invention provides the use of the aforementioned immunogenic protein/peptide polynucleotides and immune complexes in the preparation of vaccines or drugs for preventing or treating diseases caused by mutant strains of SARS-CoV-2, in one embodiment.
  • the mutant strain is a high-risk mutant strain;
  • the mutant strain is A67V, ⁇ 69-70, T95I, G142D/ ⁇ 143-145, ⁇ 211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A.
  • the strain comprises 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);
  • 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 ).
  • Another aspect of the present invention provides a modified SARS-CoV-2BA.4/BA.5ECD sequence, which includes the amino acid sequence shown in SEQ ID No: 6 or its immunogenic fragment and/or immunogenic variation. body.
  • Another aspect of the present invention provides an amino acid sequence encoding the above-mentioned immunogenic protein/peptide or an immunogenic fragment and/or immunogenic variant thereof, preferably, it is the nucleotide shown in SEQ ID No: 5 sequence or immunogenic fragments and/or immunogenic variants thereof.
  • Figure 1 is a schematic diagram of the primary structure (A) and high-level structure (B, refer to PDB: 6XLR) of the modified S-ECD.
  • Figure 2 is a diagram of the mutation sites of Omicron (BA.4/BA.5) mutant strains.
  • Figure 3 shows the purity analysis of S-Trimer-TM41C protein;
  • A Representative spectrum of non-reducing SDS-PAGE;
  • B Representative spectrum of SEC-HPLC.
  • Figure 4 shows the serum antibody titer detection (GeoMean ⁇ SD) after immunizing C57BL/6 mice with TM41 and TM41C single-component vaccines.
  • Figure 5 shows the serum antibody titer detection (GeoMean ⁇ SD) after immunizing C57BL/6 mice with SCTV01E and SCTV01E-1 vaccines.
  • antigen refers to a foreign substance that is recognized (specifically bound) by an antibody or T cell receptor, but which does not conclusively induce an immune response. Exogenous substances that induce specific immunity are called “immune antigens” or “immunogens.” "Hapten” refers to an antigen that by itself cannot elicit an immune response (although a combination of several molecular haptens, or a combination of a hapten and a macromolecular carrier can 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 an affinity to the antigens in the immunogenic composition of the invention, and a “cell-mediated immune response” is an immune response produced by T cells and /or other leukocyte-mediated immune responses.
  • a “cell-mediated immune response” is induced by the provision of antigenic epitopes associated with class I or class II molecules of the major histocompatibility complex (MHC), CD1, or other atypical MHC-like molecules.
  • MHC major histocompatibility complex
  • immunogenic composition refers to any pharmaceutical composition containing an antigen, such as a microorganism, or components thereof, which composition can be used to induce an immune response in an individual.
  • Immunogenic means that an antigen (or an epitope of an antigen), such as a coronavirus spike protein receptor binding region, or an immunogenic composition induces humoral or cell-mediated induction in a host (e.g., a mammal) The ability of an immune response or both.
  • a “protective” immune response refers to the ability of an immunogenic composition to induce a humoral or cell-mediated immune response, or both, that serves to protect an individual from infection.
  • the protection provided need not be absolute, i.e., the infection need not be completely prevented or eradicated, as 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 moderating the severity or rapid onset of symptoms of infection.
  • Immunogenic amount and “immunologically effective amount” are used interchangeably herein and refer to an antigen or immunogenic composition sufficient to elicit an immune response (cellular (T cell) or humoral (B cell or antibody) response or two). 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.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of contiguous amino acid residues.
  • nucleic acid refers to RNA, DNA, cDNA or cRNA and derivatives thereof, such as those containing modified backbones. It will be appreciated that the present invention provides polynucleotides comprising sequences complementary to sequences described herein.
  • Polynucleotide contemplated by the present invention includes the forward strand (5' to 3') and the reverse complementary strand (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.).
  • immunogenic protein/peptide includes a polypeptide that is immunologically active in the sense that it is capable of eliciting a humoral and/or cellular immune response against the protein upon administration to the host.
  • a protein fragment according to the invention comprises or essentially consists of or consists of at least one epitope or antigenic determinant.
  • an "immunogenic" protein or polypeptide includes the full-length sequence of a protein, analogs thereof, or immunogenic fragments thereof.
  • immunogenic fragment refers to a protein fragment that contains one or more epitopes, thereby triggering the above-mentioned immune response.
  • immunogenic protein/peptide also encompasses deletions, additions and substitutions to the sequence so long as the polypeptide functions to generate an immune response as defined herein, i.e. "immunogenic variants”.
  • active variants of a nucleotide sequence also encompasses deletions, additions and substitutions to the sequence, so long as the nucleotide sequence functions to generate an immune response as defined herein.
  • the SCTV01E-1 recombinant protein vaccine provided by the present invention is modified based on the extracellular domain (ECD, containing S1 and S2 parts) of the SARS-CoV-2 spike protein.
  • ECD extracellular domain
  • S1 and S2 parts the natural spike protein of SARS-CoV-2 has a trimer structure.
  • the membrane fusion process is completed through the RRAR site existing between S1 and S2, which is easily Proteases in the Golgi apparatus and on the cell surface cleave, followed by the shedding of S1, and further the S2 structure changes from the prefusion conformation to the postfusion conformation, thereby completing membrane fusion [ 20 ] .
  • the ECD trimer with a stable prefusion conformation is the next step.
  • the present invention performed the following three parts based on the S proteins of different strain variants. Transformation:
  • the present invention removes the Furin site in the SCTV01E-1 recombinant protein vaccine, that is, fixes the amino acid sequence from positions 679 to 688 as NSPGSASSVA to reduce the possibility of S1 breakage and shedding.
  • the prefusion conformation of the spike protein is unstable, and effectively inducing neutralizing antibodies requires keeping the prefusion conformation stable, which has been confirmed in RSV and HIV-1 vaccine research [ 21 , 22 ] .
  • the S-2P (i.e., mutation of amino acids 986 and 987 to proline) modification scheme is commonly used [ 23-25 ] .
  • the present invention also introduced HexaPro mutations that can effectively improve stability without affecting its three-dimensional structure (that is, in addition to the S-2P mutation, amino acids 817, 892, 899 and 942 are mutated to proline) [ 26 ] . These mutation sites are located at the N-terminal or Loop region of the ⁇ -helix in S2. Mutation to the proline (P) type with this secondary structure tendency can effectively reduce the allosteric tendency of S2 and stabilize the prefusion of S2. Conformation.
  • the present invention adds the trimerization module T4foldon to the C-terminus of the vaccine molecule.
  • This module is derived from the C-terminal domain of fiber protein of T4 bacteriophage and has 27 amino acids.
  • the corresponding trimer is prepared Proteins, namely D614G epidemic strain TM8 protein, Alpha variant strain TM22 protein, Beta variant strain TM23 protein, Delta variant strain TM28 protein, Omicron BA.1 variant strain TM41 protein and Omicron BA.4/BA.5 variant strain TM41C protein.
  • the prepared D614G epidemic strain TM8 protein vaccine was used to immunize mice and immunological assays were performed.
  • the immunological assay of the Beta variant strain TM23 protein vaccine in cynomolgus monkeys and the Alpha variant strain TM22 protein vaccine in mice were all performed. It shows that the three vaccines prepared by the present invention can produce antibody immune responses with sufficient titers in experimental animals; and the immunological results of mice using TM8+TM23 two-component vaccine and TM22+TM23 two-component vaccine (SCTV01C)
  • SCTV01C TM8+TM23 two-component vaccine
  • the evaluation also suggested that the two-component vaccine has high and similar neutralizing titers against different strains. Therefore, it has better broad-spectrum neutralizing ability than the single-component vaccine.
  • the two-component vaccine has higher and similar neutralizing potency against different mutant strains.
  • the neutralizing titer is much higher than that of the serum of recovered patients against the early epidemic strain (its genome sequence: GenBank Accession No. NC_045512).
  • Further studies have shown that the protective efficacy of the TM22+TM23+TM28+TM41 four-component vaccine (SCTV01E) is better than 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, including S1 and S2 parts) of the SARS-CoV-2 spike protein. It is a five-component vaccine TM22+TM23+TM28+TM41+TM41C.
  • the natural spike protein of SARS-CoV-2 has a trimer 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 present between S1 and S2. Open, then the shedding of S1 occurs, and the structure of S2 further changes from the prefusion conformation to the postfusion conformation, thereby completing the membrane fusion process [20] .
  • the present invention carried out the following three-part transformation based on the S protein of different strain variants (Table 1 and Figure 1):
  • the present invention removes the Furin site in the SCTV01E-1 recombinant protein vaccine, that is, fixes the amino acid sequence from positions 679 to 688 as NSPGSASSVA to reduce the possibility of S1 breakage and shedding.
  • the present invention lies in each aspect of the SCTV01E-1 recombinant protein vaccine.
  • the HexaPro mutation which can effectively improve the stability without affecting its three-dimensional structure, was introduced into the antigen component (that is, in addition to the S-2P mutation, amino acids 817, 892, 899 and 942 were mutated to proline) [ 26 ] . These mutation sites are located at the N-terminal or Loop region of the ⁇ -helix in S2. Mutation to the proline (P) type with this secondary structure tendency can effectively reduce the allosteric tendency of S2 and stabilize the prefusion of S2. Conformation.
  • the present invention adds the trimerization module T4foldon to the C-terminus of each antigen component of the vaccine molecule.
  • This module is derived from the C-terminal domain of fiber protein of T4 bacteriophage and has 27 amino acids. T4foldon has been used in RSV vaccine candidates and was proven to be safe in phase I clinical studies [ 27 ] .
  • the ECD trimer immunogenic protein/peptide of the present invention shows excellent immunogenicity in mice and can maintain long-term humoral immunity and cellular immune responses.
  • Example 1 Design of novel coronavirus recombinant spike protein extracellular domain (S-ECD) trimer protein antigen, construction of expression vector and protein production
  • T41C S-ECD trimer protein expression vector based on Omicron (BA.4/BA.5) sequence (EPI_ISL_11542270.1)
  • Figure 2 is a diagram of the mutation sites of Omicron (BA.4/BA.5) mutant strains.
  • the mutation sites include: T19I, L24del, P25del, P26del, A27S, H68del, V69del, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478 K,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-ECDTM41C-T4F-trimer.
  • the pGS5-9-A1 stable strain expression vector digested by HindIII+EcoRI was constructed by the In-fusion method to obtain the pGS5-9-CoV2-S-ECDTM41C-T4F-trimer expression vector.
  • the target gene constructed above was chemically transferred into HD-BIOP3 (GS-) cells (Horizon), cultured using self-developed serum-free medium, and stably expressed cell lines were obtained through MSX pressure screening, and cultured for 14 After three days, the culture supernatant was obtained by centrifugation and filtration.
  • the culture supernatant was first captured using cation exchange chromatography (POROS , Borgron) flow-through mode for further purification to remove impurities related to products and processes; secondly, low pH incubation and virus removal filtration (Planova) are used to inactivate and remove the virus, and finally ultrafiltration membrane bags (Millipore ) and change the ultrafiltration medium to citrate buffer.
  • the expression level of S-ECD trimer is >500mg/L.
  • Example 2 Purity and stability analysis of the new coronavirus recombinant spike protein extracellular domain (S-ECD) trimer protein
  • the above 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, pH7.0 ⁇ 0.2 , with a concentration of approximately 0.79 mg/mL, using sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the primary structure purity and size-exclusion high-performance liquid chromatography (size-exclusion) High performance liquid chromatograph, SEC-HPLC) was used to analyze its trimer content, and dynamic light scattering (DLS) was used to detect its morphological characteristics.
  • SDS-PAGE sodium dodecyl sulfonate-polyacrylamide gel electrophoresis
  • SDS-PAGE Specific steps of SDS-PAGE: (1) Preparation of SDS-PAGE gel: 3.9% stacking gel, 7.5% separating gel; (2) Boil the sample at 100°C for 2 minutes, load 8 ⁇ g after centrifugation; (3) Decolorize after staining with Coomassie Brilliant Blue .
  • SEC-HPLC operation steps are: (1) Instrument: liquid chromatography system (Agilent Company, model: Agilent1260), water-soluble size exclusion chromatography column (Sepax Company, model: SRT-C SEC-500 column); (2 )Mobile phase: 200mM NaH 2 PO 4 , 100mM Arginine, pH 6.5, 0.01% isopropanol (IPA); (3) Sample loading volume is 80 ⁇ g; (3) Detection wavelength is 280nM, analysis time is 35min, flow rate is 0.15mL /min.
  • the recombinant TM41C protein has a homotrimeric structure due to its non-covalent hydrophobic interaction. After non-reducing SDS-PAGE treatment, it became a monomer molecule with a molecular weight of about 148KDa ( Figure 3), with a purity of 95.2%; SEC-HPLC showed that the purity of the trimer was 97.2%, and the ratio of aggregates to fragments was less than 5 %, the average molecular weight of its main peak is 512KDa; the dynamic light scattering results show that the average radius of the TM41C trimer protein molecule is 8.1nm (Table 2).
  • the recombinant TM41C trimer protein was stored at 37°C for 1 week (37T1W), stored at -80°C for 8 hours, and then transferred to 45°C for 0.5 hours (F/T) to thaw for 5 times.
  • SDS-PAGE and SEC-HPLC analyzed the changes in trimer content. The data are shown in Table 3.
  • Example 3 TM41 single-component vaccine and multi-component vaccine in Immunological evaluation of mice
  • TM22 and TM23 trimer proteins For the expression and purification of TM22 and TM23 trimer proteins, please refer to the patent "A method to improve the immunogenicity of SARS-CoV-2 mutant strain ECD antigen/antigen trimer stability" (Application No.: PCT/CN2022/095609 and Its priority application numbers: 202110606512.2 and 202111237604.4, the full text of which is incorporated herein).
  • the applicant elaborated in the patent that the two-component vaccine composed of TM22+TM23 has better broad-spectrum neutralizing ability than the TM22 and TM23 single-component vaccines.
  • TM28 trimer protein For the expression and purification of TM28 trimer protein, please refer to the patent "Preparation and application of a recombinant multi-component novel coronavirus trimer protein vaccine that can induce broad-spectrum neutralizing activity" (Application No.: PCT/CN2022/107213 and its priority Application number: 202110838359.6 is incorporated in its entirety here).
  • the applicant elaborated in the patent application that the trivalent vaccine composed of TM22+TM23+TM28 has better broad-spectrum neutralizing ability than the single-component vaccine.
  • TM41 trimer protein For the expression and purification of TM41 trimer protein, please refer to the patent application "Preparation and application of a recombinant multi-component novel coronavirus trimer protein vaccine that can induce broad-spectrum neutralizing activity" (application number: PCT/CN2023/078135 and its priority Rights application number: 202210184528.3 is incorporated in its entirety here).
  • application number: PCT/CN2023/078135 and its priority Rights application number: 202210184528.3 is incorporated in its entirety here.
  • the applicant elaborated in the patent application that the quadrivalent vaccine composed of TM22+TM23+TM28+TM41 has better broad-spectrum neutralizing ability than the single-component vaccine.
  • the applicant added the TM41C component to the TM22+TM23+TM28+TM41 quadrivalent vaccine to form a five-part vaccine.
  • Component vaccines In order to further expand the broad-spectrum neutralizing effect of the vaccine, especially the neutralizing effect of the new Omicron subtype variant strain, the applicant added the TM41C component to the TM22+TM23+TM28+TM41 quadrivalent vaccine to form a five-part vaccine. Component vaccines.
  • the purified TM22, TM23, TM28, TM41 and TM41C trimer proteins were pre-diluted with PBS and then equal volumes with MF59 (8 ⁇ , source: China Cell Engineering Co., Ltd., the same below) Mix and prepare single-component or multi-component vaccine samples.
  • mice Female C57BL/6 mice aged 6-8 weeks (source: Beijing Vitong Lever Laboratory Animal Technology Co., Ltd., weight 18-20g) were injected intramuscularly with 0.1 mL of vaccine sample containing MF59 adjuvant. A total of 2 immunizations were carried out, with an interval of 14 days. Blood was collected from the orbit 14 days after the first immunization (14 days after the first immunization) and 7 days after the second immunization (7 days after the second immunization). The serum was collected by centrifugation at 4500 rpm for 15 minutes for subsequent serological immune analysis.
  • Omicron a replication-deficient vesicular stomatitis virus in which the VSV-G protein gene in the viral genome is replaced with a luciferase reporter gene (i.e., VSV ⁇ G-Luc-G ) is a vector, amplified and prepared in cell lines expressing Spike and its mutant proteins, prepared by China Cell Engineering Co., Ltd., the same below), mixed and incubated in a 37°C, 5% CO 2 incubator for 1 hour.
  • pseudovirus pseudovirus is a replication-deficient vesicular stomatitis virus in which the VSV-G protein gene in the viral genome is replaced with a luciferase reporter gene (i.e., VSV ⁇ G-Luc-G ) is a vector, amplified and prepared in cell lines expressing Spike and its mutant proteins, prepared by China Cell Engineering Co., Ltd., the same below), mixed and incubated in a 37°C, 5% CO 2 incubator for 1 hour.
  • the cell wells containing pseudovirus without serum were used as positive controls, and the cell wells without serum and pseudovirus were used as negative controls.
  • 2 ⁇ 10 4 Huh-7 cells were inoculated at 100 ⁇ L/well, mixed and placed in a 37°C, 5% CO 2 incubator for about 20 hours.
  • Neutralization rate % (positive control RLUs – sample RLUs) / (positive control RLUs – negative control RLUs) ⁇ 100%. Calculate IC 50 according to the Reed-Muench formula, which is the neutralizing potency NAT 50 .
  • the results of serum neutralizing antibody detection after 2 and 7 days of vaccination are shown in Figure 4.
  • the TM41 single-component vaccine induced the neutralization of Omicron BA.1, BA.2, BA.2.12.1, BA.2.75 and BA.4/5.
  • the antibody titers (geometric mean NAT 50 ) were 17277, 2520, 873, 72 and 100 respectively.
  • the NAT 50 induced by the TM41C single-component vaccine against the above mutant strains were 108, 4319, 7678, 1433 and 33518 respectively.
  • the NAT 50 induced by the 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 and 8.7 times that induced by the TM41 single component vaccine respectively. times, 14.3 times and 493.4 times. This shows that compared with the TM41 single-component vaccine, the TM41C single-component vaccine has a broader spectrum of neutralizing activity against new Omicron subtype variants.
  • the NAT 50 induced by SCTV01E vaccine against Omicron BA.1, BA.2.12.1, BA.2.75 and BA.4/5 was 4633, 543, 207 and 123 respectively.
  • the NAT 50 induced by SCTV01E-1 vaccine against the above mutant strains The NAT 50 induced by SCTV01E-1 vaccine against Omicron BA.1, BA.2.12.1, BA.2.75 and BA.4/5 was 0.3 times and 12.5 times that induced by SCTV01E vaccine, respectively. , 12.0 times and 131.6 times ( Figure 5).
  • Figure 5 is the results of serum antibody titer detection (GeoMean ⁇ SD) after immunizing C57BL/6 mice with SCTV01E and SCTV01E-1 vaccines.
  • the five-component vaccine has a broad-spectrum neutralizing ability against new Omicron subtype mutant strains. Because it covers more mutation sites, it is expected to have cross-protective capabilities against multiple mutant strains and improve the resistance to mutations. Protection rate against strain infection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Communicable Diseases (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明涉及分子疫苗学领域，提供了一种可诱导广谱中和活性的重组多组分新冠病毒三聚体蛋白疫苗。重组蛋白成分包含但不限于Alpha(B.1.1.7)、Beta(B.1.351)、Delta(B.1.617.2)和Omicron(BA.1、BA.4/BA.5)刺突蛋白(S蛋白)的胞外结构域(ECD)通过引入突变位点和三聚化辅助结构形成的同源三聚体蛋白。多组分疫苗包含上述变异株的单一成分或任意组合成分的ECD三聚体蛋白以及药学上可接受的佐剂。该疫苗组合在小鼠显示出优异的免疫原性，可维持长时程的体液免疫和细胞免疫反应。所述多组分新冠病毒三聚体蛋白疫苗可用于预防由新冠病毒及其变异株感染引发的感染相关疾病。

Description

一种可诱导广谱中和活性重组五组分新冠病毒三聚体蛋白疫苗的制备及应用
相关申请的交叉引用
本申请要求2022年08月08日提交的中国专利申请202210946805.X的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及分子疫苗学领域，涉及一种可诱导广谱中和活性重组多组分新冠病毒三聚体蛋白疫苗的制备及应用。
背景技术
新型冠状病毒(SARS-CoV-2)具有较强的传播能力，安全有效的疫苗是控制疫情的最有力的技术手段。根据靶点和技术的不同，疫苗可以被分为以下几类：灭活疫苗、重组蛋白疫苗、病毒载体疫苗、RNA疫苗、减毒活疫苗和病毒样颗粒疫苗等。自SARS-CoV-2大流行以来，各国研制的新冠疫苗已达200多个。截止到2022年8月03日，全球已有40种疫苗已被批准使用或附条件使用，另外已有210种疫苗进入临床研究(https://covid19.trackvaccines.org/vaccines/)。
SARS-CoV-2和SARS-CoV具有共同的宿主细胞受体蛋白，即血管紧张素转化酶2(ACE2)[1]。病毒的三聚体刺突蛋白(Spike)同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽[2]。S蛋白是病毒包膜的主要成分，在受体结合，融合，病毒进入和宿主免疫防御方面具有重要的作用。S蛋白的RBD区含有主要的中和抗体表位，可刺激B细胞产生针对RBD的高滴度中和抗体。此外，S蛋白还含有丰富的T细胞表位，可诱导T细胞发生特异性CTL反应，清除病毒感染的细胞。因此，S蛋白是新冠疫苗设计的最关键抗原。目前设计的绝大多数疫苗都选择了S蛋白或RBD结构域蛋白作为核心免疫原。
SARS-CoV-2为RNA单链病毒，易发生缺失突变且这种突变多发生在S蛋白的重复缺失区(Recurrent deletion regions,RDRs)。缺失或突变可能改变S蛋白的构象，使得先前疫苗免疫诱导的抗体降低对突变S蛋白的结合和中和而导致疫苗免疫效果的下降和病毒的免疫逃逸。早期的D614G突变(B.1)增强了S蛋白同ACE2受体的亲和力，并迅速成为了流行株，但该突变未降低对中和抗体的敏感性[ 3 , 4 ]。然而，随着SARS-CoV-2的大流行，全球出现了5种高关注变异株(Variants of Concern,VOC)：Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Delta(B.1.617.2)和Omicron(B.1.1.529)以及2种需留意变异株(Variants of Interest,VOI)：Lambda(C.37)和Mu(B.1.621)。研究表明这些高风险毒株可增加传播性、加重疾病发展(增加住院治疗或死亡率)、严重降低既往感染或免疫接种所产生的抗体中和作用、降低治疗或疫苗效用降低或使诊断检测失效[ 5 ]。Alpha传播迅速，且可增加61％相关死亡风险[ 6 ]。中和效应研究结果表明，康复者血浆或疫苗免疫者血清对Alpha的中和能力基本保持不变，然而对Beta的中和能力却大幅下降[ 7-12 ]。临床结果也表明，Alpha对疫苗的保护效果影响不大，而Beta则会大幅降低对轻症的保护效果[ 13-16 ]。相比原始病毒和早期变异毒株，Delta变异的传播力更强，潜伏期短，发病进程快，还能降低疫苗的保护作用。
SARS-CoV-2的Omicron谱系已进化出多个亚型。BA.1在最初的感染中占主导地位，但全球范围内很快被BA.2取代。2022年4月上旬，报告了两个新的Omicron谱系，分别命名为BA.4和BA.5。BA.4和BA.5包含相同的S序列，虽然与BA.2密切相关，但它们在其RBD结构域中包更多的突变[17]。BA.4/BA.5具有更强的逃逸中和抗体能力表明BA.4/BA.5比BA.1或BA.2更容易在疫苗接种者或突破感染的人群中传播[17-19]。BA.4/BA.5目前已经成为全球主要的流行株。目前的疫苗均是基于早期流行株(其基因组序列：GenBank Accession No.NC_045512)序列进行的设计，鉴于Alpha、Delta、Omicron新亚型的高传播性以及Beta和Omicron对现有疫苗保护效力的不利影响，迫切需要对高风险变异体具有高保护效力的多组分新型疫苗。
发明内容
基于以上对于新型冠状病毒SARS-CoV-2变异株具有高保护效果的疫苗的需求，
本发明的第一方面提供提高SARS-CoV-2突变毒株ECD抗原免疫原性/抗原三聚体稳定性的方法，该方法通过构建包含SEQ ID No:8所示的氨基酸序列,或其免疫原性片段和/或免疫原性变体的ECD抗原，从而
ECD为稳定的prefusion构象的三聚体形式；
优选地，突变毒株为含有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之至少任一的高风险突变毒株；
在一个实施方案中，该毒株为Omicron(BA.4/BA.5)；
在一个实施方案中，该ECD抗原和选自以下的一种或多种佐剂共同施予受试者：
铝佐剂、油乳佐剂、Toll样受体(TLR)激动剂、免疫增强剂的组合、微生物类佐剂、蜂胶佐剂、左旋咪唑佐剂、脂质体佐剂、中药佐剂及小肽类佐剂；
在一个实施方案中，油乳佐剂包含角鲨烯成分；
Toll样受体(TLR)激动剂包含吸附在铝盐上的CpG或单磷酰脂质A(MPL))；和
免疫增强剂的组合包含QS-21和/或MPL。
本发明的另一方面提供一种提高SARS-CoV-2突变毒株ECD抗原免疫原性//抗原三聚体稳定性的方法，该方法通过
构建编码包含SEQ ID No:8所示的氨基酸序列,或其免疫原性片段和/或免疫原性变体的多核苷酸，
从而表达稳定的prefusion构象的三聚体形式ECD；
在一个实施方案中，突变毒株为含有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和N969K之至少任一的高风险突变毒株；
在一个实施方案中，该毒株为Omicron(BA.4/BA.5)，
在一个实施方案中，构建包含SEQ ID No:7所示的核苷酸序列或其片段的多核苷酸。
本发明的另一方面提供一种免疫原性/抗原三聚体稳定性提高的SARS-CoV-2突变毒株ECD免疫原性蛋白/肽，其特征在于，该免疫原性蛋白/肽包含SEQ ID No:8所示的氨基酸序列,或其免疫原性片段和/或免疫原性变体，
该ECD免疫原性蛋白/肽为稳定的prefusion构象的三聚体形式；
在一个实施方案中，突变毒株为含有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和N969K之至少任一的高风险突变毒株；
在一个实施方案中,该毒株为Omicron(BA.4/BA.5)。
本发明的另一方面提供一种多核苷酸，其编码如前所述的免疫原性蛋白/肽，
在一个实施方案中，包含SEQ ID No:7所示的核苷酸序列。
本发明的另一方面提供一种免疫原性组合物，其特征在于，包含
如前所述的免疫原性蛋白/肽，或
如前所述的多核苷酸，和
药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合；
任选地，包含佐剂。
本发明的另一方面提供多价免疫原性组合物，其特征在于，进一步包含
SEQ ID No:16、SEQ ID No:20、SEQ ID No:28或SEQ ID No:32至少之任一所示的氨基酸序列,或其免疫原性片段和/或免疫原性变体，或
SEQ ID No:15、SEQ ID No:19、SEQ ID No:27或SEQ ID No:31至少之任一所示的核苷酸序列或其活性变体。
在一个实施方案中，免疫原性组合物的佐剂选自以下的一种或多种：
铝佐剂、油乳佐剂、Toll样受体(TLR)激动剂、免疫增强剂的组合、微生物类佐剂、蜂胶佐剂、左旋咪唑佐剂、脂质体佐剂、中药佐剂及小肽类佐剂；
在一个实施方案中，油乳佐剂包含角鲨烯成分；
Toll样受体(TLR)激动剂包含吸附在铝盐上的CpG或单磷酰脂质A(MPL))；和
免疫增强剂的组合包含QS-21和/或MPL。
本发明的另一方面提供前述免疫原性蛋白/肽、多核苷酸和免疫复合物预防或治疗SARS-CoV-2突变毒株引起的疾病的用途，优选地，突变毒株为高风险突变毒株；
在一个实施方案中，突变毒株为含有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和N969K之至少任一的高风险突变毒株；
在一个实施方案中，该毒株包含D614G突变(B.1)、Beta(B.1.351)、Alpha(B.1.1.7)、Delta(B.1.617.2)、P.1、B.1.427，B.1.429和/或Omicron(BA.1、BA.4/BA.5)；
在一个实施方案中，该毒株包含Alpha(B.1.1.7)、Beta(B.1.351)、Delta(B.1.617.2)和Omicron(BA.1、BA.4/BA.5)。
本发明的另一方面提供前述的免疫原性蛋白/肽多核苷酸和免疫复合物在制备预防或治疗SARS-CoV-2突变毒株引起的疾病的疫苗或药物中用途，在一个实施方案中，突变毒株为高风险突变毒株；
在一个实施方案中，突变毒株为含有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和N969K之至少任一的高风险突变毒株；
在一个实施方案中，该毒株包含D614G突变(B.1)、Beta(B.1.351)、Alpha(B.1.1.7)、Delta(B.1.617.2)P.1、B.1.427，B.1.429和/或Omicron(BA.1、BA.4/BA.5)；
在一个实施方案中，该毒株包含Alpha(B.1.1.7)、Beta(B.1.351)、Delta(B.1.617.2)和/或Omicron(BA.1、BA.4/BA.5)。
本发明的另一方面提供一种改造过的SARS-CoV-2BA.4/BA.5ECD序列，其包含SEQ ID No:6所示的氨基酸序列或其免疫原性片段和/或免疫原性变体。
本发明的另一方面提供一种编码上述免疫原性蛋白/肽氨基酸序列或其免疫原性片段和/或免疫原性变体，优选地，其为SEQ ID No:5所示的核苷酸序列或其免疫原性片段和/或免疫原性变体。
附图说明
图1为经过改造的S-ECD的一级结构(A)及高级结构(B，参考PDB:6XLR)的示意图。
图2为Omicron(BA.4/BA.5)变异株突变位点图示。
图3为S-Trimer-TM41C蛋白纯度分析；(A)非还原SDS-PAGE代表性图谱；(B)SEC-HPLC代表性图谱。
图4示出了TM41和TM41C单组分疫苗免疫C57BL/6小鼠后血清抗体效价检测(GeoMean±SD)。
图5示出SCTV01E和SCTV01E-1疫苗免疫C57BL/6小鼠后血清抗体效价检测(GeoMean±SD)。
具体实施方式
定义
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为了本发明的目的，进一步定义以下术语。
当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。
术语“包括”、“包含”是指包括具体成分而不排除任何其他的成分。诸如“基本上由……组成”允许包括不损害本发明的新颖或基本特征的其他成分或步骤，即，它们排除损害本发明的新颖或基本的特征的其他未列举的成分或步骤。术语“由……组成”是指包括具体成分或成分组并且排除所有其他成分。
术语“抗原”是指一种由抗体或T细胞受体所识别(特异性结合)的外源物质，但是其不能确定性地诱导免疫应答。诱导特异性免疫的外源性物质称为“免疫性抗原”或“免疫原”。“半抗原”是指一种本身不能引发免疫应答(尽管几个分子半抗原的结合物，或半抗原与大分子载体的结合物可引发免疫应答)的抗原。
“体液免疫应答”是抗体介导的免疫应答并且涉及引入和生成以一定亲和力识别和结合本发明的免疫原性组合物中的抗原的抗体，“细胞介导的免疫应答”是由T细胞和/或其他白细胞介导的免疫应答。“细胞介导的免疫应答”是通过提供与主要组织相容性复合物(MHC)的I类或II类分子、CD1或其他非典型MHC样分子相关的抗原表位而诱发的。
术语“免疫原性组合物”是指含有抗原如微生物或其组分的任何药物组合物，该组合物可用于在个体中诱发免疫应答。
如本文所使用的“免疫原性”意指抗原(或抗原的表位)例如冠状病毒棘突蛋白受体结合区或免疫原性组合物在宿主(例如哺乳动物)中诱发体液或细胞介导的免疫应答或二者的能力。
“保护性”免疫应答是指免疫原性组合物诱发用于保护个体免于感染的体液或细胞介导的免疫应答或两者的能力。所提供的保护不必是绝对的，即，不必完全阻止或根除感染，只要相对于对照个体群体(例如未给药疫苗或免疫原性组合物的受感染动物)存在统计学上显著的改进即可。保护可限于缓和感染症状的严重性或发作快速性。
“免疫原性量”和“免疫有效量”二者在本文可交换使用，是指抗原或免疫原性组合物足以引发免疫应答(细胞(T细胞)或体液(B细胞或抗体)应答或二者，如通过本领域技术人员已知的标准测定所测量的)的量。
抗原作为免疫原的有效性可通过例如增殖测定、通过细胞溶解测定、或通过测量B细胞活性水平来测量。
术语“多肽”和“蛋白质”在本文中可互换使用，指连续氨基酸残基的聚合物。
术语“核酸”、“核苷酸”和“多核苷酸”可互换使用，是指RNA、DNA、cDNA或cRNA及其衍生物，诸如含有经修饰的骨架的那些。应当理解，本发明提供了包含与本文所述序列互补的序列的多核苷酸。本发明中考虑的“多核苷酸”包括正向链(5'至3')和反向互补链(3'至5')。根据本发明的多核苷酸可以以不同方式(例如通过化学合成，通过基因克隆等)制备，并且可采取各种形式(例如直链或支链的，单链或双链的，或其杂合体，引物，探针等)。
术语“免疫原性蛋白/肽”包括在一旦向宿主施用，其能够引发针对该蛋白质的体液和/或细胞类型的免疫反应的意义上，具有免疫活性的多肽。因此，根据本发明的蛋白质片段包含至少一个表位或抗原决定簇或基本上由其组成或由其组成。如本文中所用，“免疫原性”蛋白质或多肽包括蛋白质的全长序列、其类似物或其免疫原性片段。“免疫原性片段”是指包含一个或多个表位，从而引发上述免疫反应的蛋白质片段。
术语“免疫原性蛋白/肽”还涵盖了对序列的缺失、添加和取代，只要该多肽起到产生如本文所定义的免疫反应的作用即可，即“免疫原性变体”。
术语“核苷酸序列的活性变体”还涵盖了对序列的缺失、添加和取代，只要该核苷酸序列起到产生如本文所定义的免疫反应的作用即可。
本发明提供的SCTV01E-1重组蛋白疫苗，是基于SARS-CoV-2刺突蛋白的胞外结构域(ECD，含S1和S2部分)改造而来。已知的SARS-CoV-2的天然刺突蛋白为三聚体结构，在其产生和行使侵染功能的过程中，膜融合过程的完成是通过S1和S2间存在的RRAR位点而易被高尔基体中以及细胞表面的蛋白酶切开，随后发生S1的脱落，进一步地S2结构由prefusion构象转变为postfusion构象，从而完成膜融合[ 20 ]。基于此，具有prefusion的稳定构象的ECD三聚体，是进行下一步，为了获得稳定的prefusion构象的ECD三聚体，本发明在不同毒株变体的S蛋白基础上，进行了如下三部分改造：
目前发现，具有较高中和活性的抗体都结合于S1区域(具体来说结合于S1中的NTD和RBD区域)。保持S1部分的完整，对于新冠疫苗诱导中和抗体的产生至关重要。本发明在SCTV01E-1重组蛋白疫苗中改造去除了Furin位点，即将679至688位的氨基酸序列固定为NSPGSASSVA，以降低S1断裂与脱落的可能性。
2)由于S2自身的变构倾向，使得刺突蛋白的prefusion构象不稳定，而有效地诱发中和抗体需要保持prefusion构象稳定，这在RSV和HIV-1疫苗研究中已经被证实[ 21 , 22 ]。当前的上市疫苗中，普遍采用了S-2P(即将986和987位氨基酸突变为脯氨酸)改造方案[ 23-25 ]。此外，本发明还引入了能有效提升稳定性且不影响其三维结构的HexaPro突变(即除了S-2P突变外，又将817，892，899和942位氨基酸突变为脯氨酸)[ 26 ]。这些突变位点都位于S2中的α-螺旋N端或Loop区，突变为具有该二级结构倾向的脯氨酸(P)类型后，可以有效的降低S2的变构倾向从而稳定S2的prefusion构象。
3)最后，为了进一步稳定S-ECD三聚体结构，本发明在疫苗分子的C端加入了三聚化模块T4foldon。该模块来源于T4噬菌体的纤维蛋白的C端结构域，具有27个氨基酸。
在使用经上述改造后的重组S-ECD三聚体蛋白抗原重组进表达载体、并对表达出的重组S-ECD三聚体蛋白进行常规纯度和稳定性分析后，制备成相应的三聚体蛋白，即D614G流行株TM8蛋白、Alpha变异株TM22蛋白、Beta变异株TM23蛋白、Delta变异株TM28蛋白、Omicron BA.1变异株TM41蛋白和Omicron BA.4/BA.5变异株TM41C蛋白。
使用制备出的D614G流行株TM8蛋白疫苗免疫小鼠后进行免疫学测定、Beta变异株TM23蛋白疫苗在食蟹猴中的免疫学测定以及Alpha变异株TM22蛋白疫苗在小鼠中的免疫学测定均显示本发明制备的这三种疫苗能够在实验动物体内产生足够效价的抗体免疫反应；并且在使用TM8+TM23双组分疫苗以及TM22+TM23双组分疫苗(SCTV01C)在小鼠的免疫学评价中也提示，双组分疫苗对不同毒株均具有较高且相近中和效价，因此相比单组分疫苗具有更优的广谱中和能力，双组分疫苗对不同变异株的中和效价远高于康复者血清对早期流行株(其基因组序列：GenBank Accession No.NC_045512)的中和效价。进一步研究表明，在保护Delta和Omicron变异株感染时，TM22+TM23+TM28+TM41四组分疫苗(SCTV01E)的保护效力优于TM22+TM23双组分疫苗。
SCTV01E-1重组蛋白疫苗，基于SARS-CoV-2刺突蛋白的胞外结构域(ECD，含S1和S2部分)改造而来，为TM22+TM23+TM28+TM41+TM41C五组分疫苗。SARS-CoV-2的天然刺突蛋白为三聚体结构，在其产生和行使侵染功能的过程中，由于S1和S2间存在的RRAR位点而易被高尔基体中以及细胞表面的蛋白酶切开，随后发生S1的脱落，进一步地S2结构由prefusion构象转变为postfusion构象，从而完成膜融合过程[20]。为了获得稳定的prefusion构象的ECD三聚体，本发明在不同毒株变体的S蛋白基础上，进行了如下三部分改造(表1和图1)：
1)目前发现，具有较高中和活性的抗体都结合于S1区域(具体来说结合于S1中的NTD和RBD区域)。保持S1部分的完整，对于新冠疫苗诱导中和抗体的产生至关重要。本发明在SCTV01E-1重组蛋白疫苗中改造去除了Furin位点，即将679至688位的氨基酸序列固定为NSPGSASSVA，以降低S1断裂与脱落的可能性。
2)由于S2自身的变构倾向，使得刺突蛋白的prefusion构象不稳定，而有效地诱发中和抗体需要保持prefusion构象稳定，这在RSV和HIV-1疫苗研究中已经被证实[ 21 , 22 ]。目前处于上市或临床阶段的新冠疫苗，大多数聚焦于对病毒侵染发挥重要功能的刺突蛋白部分，因此如何保证其稳定在prefusion构象成为关注点。在当前的上市疫苗中，普遍采用了S-2P(即将986和987位氨基酸突变为脯氨酸)改造方案[ 23-25 ]。为了进一步提升ECD的prefusion构象稳定性，以提升其在CHO重组细胞中的表达量和产品稳定性，使其在降低生产成本的同时便于存储和运输，本发明在于SCTV01E-1重组蛋白疫苗的各个抗原组分中引入了能有效提升稳定性且不影响其三维结构的HexaPro突变(即除了S-2P突变外，又将817，892，899和942位氨基酸突变为脯氨酸)[ 26 ]。这些突变位点都位于S2中的α-螺旋N端或Loop区，突变为具有该二级结构倾向的脯氨酸(P)类型后，可以有效的降低S2的变构倾向从而稳定S2的prefusion构象。
3)最后，为了进一步稳定S-ECD三聚体结构，本发明在疫苗分子各抗原组分的C端加入了三聚化模块T4foldon。该模块来源于T4噬菌体的纤维蛋白的C端结构域，具有27个氨基酸。T4foldon曾被用于过RSV候选疫苗中，并在临床I期研究中被证明安全性良好[ 27 ]。
表1 SCTV01E-1疫苗各抗原组分的分子结构设计改造
本发明的ECD三聚体免疫原性蛋白/肽在小鼠中显示出优异的免疫原性，可维持长时程的体液免疫和细胞免疫反应。
实施例
实施例1：新冠病毒重组刺突蛋白胞外区(S-ECD)三聚体蛋白抗原设计、表达载体的构建及蛋白生产
1.1基于Omicron(BA.4/BA.5)序列(EPI_ISL_11542270.1)的S-ECD三聚体蛋白(TM41C)表达载体的构建
图2为Omicron(BA.4/BA.5)变异株突变位点图示。其突变位点包括：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包含3693bp的基因片段，通过PCR从模板pSE-CoV2-S-ECDTM41C-T4F-trimer获得TM41C基因片段。通过In-fusion方法构建到HindIII+EcoRI酶切的pGS5-9-A1稳定株表达载体中，获得pGS5-9-CoV2-S-ECDTM41C-T4F-trimer表达载体。
扩增引物
1.2 TM41C三聚体蛋白的表达和纯化
将上述构建的目的基因通过化学法转入到HD-BIOP3(GS-)细胞中(Horizon),采用自主研发的无血清培养基培养，经过MSX加压筛选获得稳定表达的细胞株，加料培养14天后，经过离心和过滤获得培养上清液。培养上清液首先采用阳离子交换层析(POROS XS，Thermo)捕获，用高盐缓冲液进行洗脱；然后采用阴离子层析(NanoGel-50Q，NanoMicro)结合模式和混合阴离子层析(DiamondMIX-A，博格隆)流穿模式进行进一步的精纯，去除与产品和工艺相关杂质；其次采用低pH孵育和除病毒过滤(Planova)对病毒进行灭活和去除，最后用超滤膜包(Millipore)进行超滤换液至柠檬酸盐缓冲液。 S-ECD三聚体表达水平>500mg/L。
实施例2：新冠病毒重组刺突蛋白胞外区(S-ECD)三聚体蛋白纯度及稳定性分析
2.1重组TM41C三聚体蛋白纯度分析
将上述纯化后重组S-ECD三聚体蛋白原液置于含1.7mM枸橼酸，8mM枸橼酸钠，300mM氯化钠，0.3g/kg聚山梨酯80，pH7.0±0.2缓冲液中，浓度约0.79mg/mL，应用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS polyacrylamide gel electrophoresis，SDS-PAGE)分析一级结构纯度和分子排阻高效液相色谱(size-exclusion high performance liquid chromatograph，SEC-HPLC)分析其三聚体含量，应用动态光散射(dynamic light scattering，DLS)检测其形态学特征。
SDS-PAGE具体操作步骤：(1)SDS-PAGE胶配制：3.9％浓缩胶，7.5％分离胶；(2)样品100℃煮沸2min，离心后上样8μg；(3)考马斯亮蓝染色后脱色。SEC-HPLC操作步骤为：(1)仪器：液相色谱系统(Agilent公司，型号：Agilent1260)，水溶性体积排阻色谱柱(Sepax公司，型号：SRT-C SEC-500色谱柱)；(2)流动相：200mM NaH2PO4，100mM Arginine，pH 6.5，0.01％异丙醇(IPA)；(3)上样量为80μg；(3)检测波长280nM，分析时间为35min，流速为0.15mL/min。
DLS具体操作步骤：(1)仪器：动态光散射仪(Wyatt Technology公司，型号：DynaPro NanoStar)；(2)上样量为50μL；(3)采集数据后，应用Dynamics 7.1.8软件分析数据。
重组TM41C蛋白由于其非共价疏水作用为同源三聚体结构。经非还原SDS-PAGE处理后成为分子量大小约148KDa的单体分子(图3)，纯度为95.2％；SEC-HPLC显示三聚体纯度为97.2％，其聚集体与片段比例含量均低于5％，其主峰分子量平均为512KDa；动态光散射结果显示TM41C三聚体蛋白分子平均半径为8.1nm(表2)。
表2重组S-ECD三聚体纯度分析
2.2重组TM41C三聚体蛋白稳定性评价
将重组TM41C三聚体蛋白分别置于37℃中保存1周(37T1W)，-80℃条件保存8h后转移至45℃条件解冻0.5h(F/T)，如此进行5次反复冻融，应用SDS-PAGE、SEC-HPLC分析其三聚体含量变化，数据见表3。
结果如表3所示，重组TM41C三聚体蛋白37℃加速1周后和5次反复冻融后，非还原SDS-PAGE纯度与SEC-HPLC三聚体含量均在88.0％以上，加速后纯度变化在2.0％以内,聚集体与片段无显著增加，表现出了良好的热加速稳定性和冻融稳定性。
表3重组TM41C三聚体蛋白稳定性评价
实施例3：TM41单组分疫苗及多组分疫苗在小鼠的免疫学评价
3.1疫苗制备及免疫方案
TM22和TM23三聚体蛋白的表达及纯化参见专利《一种提高SARS-CoV-2突变毒株ECD抗原免疫原性/抗原三聚体稳定性的方法》(申请号：PCT/CN2022/095609以及其优先权申请号：202110606512.2及202111237604.4，在此全文引入)。申请人在该专利中详细阐述了TM22+TM23组成的双组分苗相比TM22以及TM23单组分疫苗具有更优秀的广谱中和能力。TM28三聚体蛋白的表达及纯化参见专利《一种可诱导广谱中和活性重组多组分新冠病毒三聚体蛋白疫苗的制备及应用》(申请号：PCT/CN2022/107213以及其优先权申请号：202110838359.6在此全文引入)。申请人在该专利申请中详细阐述了TM22+TM23+TM28组成的三价苗相比单组分疫苗具有更优秀的广谱中和能力。TM41三聚体蛋白的表达及纯化参见专利申请《一种可诱导广谱中和活性重组多组分新冠病毒三聚体蛋白疫苗的制备及应用》(申请号：PCT/CN2023/078135以及其优先权申请号：202210184528.3在此全文引入)。申请人在该专利申请中详细阐述了TM22+TM23+TM28+TM41组成的四价苗相比单组分疫苗具有更优秀的广谱中和能力。为进一步扩宽疫苗的广谱中和效果，特别是针对Omicron新亚型变异株的中和效果，申请人在TM22+TM23+TM28+TM41四价苗的基础上加入了TM41C成分，组成了五组分疫苗。
根据最终免疫剂量(表4)将纯化获得的TM22、TM23、TM28、TM41和TM41C三聚体蛋白用PBS进行预稀释后与MF59(8×，来源：神州细胞工程有限公司，下文同)等体积混合制备单组分或多组分疫苗样品。
表4免疫分组信息汇总

3.2小鼠免疫
6-8周雌性C57BL/6小鼠(来源：北京维通利华实验动物技术有限公司，体重18-20g)，肌肉注射0.1mL含MF59佐剂的疫苗样品。共进行2次免疫，免疫间隔为14天。首次免疫14天(1免14天)及2次免疫后7天(2免7天)进行眼眶采血，4500rpm离心15分钟取血清，进行后续血清学免疫分析。
3.3小鼠免疫血清中和效价的测定
将不同稀释倍数的2免7天免疫血清50μL/孔加入96孔板，然后50μL/孔加入100～200TCID50的Omicron(BA.1)、Omicron(BA.2.12.1)、Omicron(BA.2.75)或Omicron(BA.4/5)假病毒(假病毒是以病毒基因组中VSV-G蛋白基因替换为荧光素酶报告基因的复制缺陷型水疱性口炎病毒(即VSV△G-Luc-G)为载体，在表达Spike及其突变体蛋白的细胞系中进行扩增制备，由神州细胞工程有限公司制备，下文同)，混匀后置于37℃、5％CO2培养箱孵育1h。以加入假病毒不含血清的细胞孔作为阳性对照，以不含血清和假病毒的细胞孔为阴性对照。孵育结束后，100μL/孔接种2×104个Huh-7细胞，混匀后置于37℃、5％CO2培养箱中静置培养约20h。培养结束后，去掉培养上清，50μL/孔加入1×Passive lysis buffer，混匀裂解细胞。取40μL/孔转入96孔全白化学发光板，采用LB960微孔板式发光检测仪40μL/孔加入荧光素酶底物并检测发光值(RLU)，计算中和率。中和率％＝(阳性对照RLUs–样品RLUs)/(阳性对照RLUs–阴性对照RLUs)×100％，根据Reed-Muench公式计算IC50，即为中和效价NAT50。
2免7天血清中和抗体检测结果如图4所示，TM41单组分疫苗诱导对Omicron BA.1、BA.2、BA.2.12.1、BA.2.75和BA.4/5的中和抗体滴度(几何平均值NAT50)分别为17277、2520、873、72和100，TM41C单组分疫苗诱导对上述变异毒株的NAT50分别为108、4319、7678、1433和33518，TM41C单组分疫苗诱导对Omicron BA.1、BA.2、BA.2.12.1、BA.2.75和BA.4/5的NAT50分别是TM41单组分疫苗诱导的1/159.9倍、1.7倍、8.7倍、14.3倍和493.4倍。说明与TM41单组分疫苗相比，TM41C单组分疫苗对Omicron新亚型变异株具有更广谱的中和活性。
SCTV01E疫苗诱导对Omicron BA.1、BA.2.12.1、BA.2.75和BA.4/5的NAT50分别为4633、543、207和123，SCTV01E-1疫苗诱导对上述变异毒株的NAT50分别为1243、6789、2498和16194，SCTV01E-1疫苗诱导对Omicron BA.1、BA.2.12.1、BA.2.75和BA.4/5的NAT50分别是SCTV01E疫苗诱导的0.3倍、12.5倍、12.0倍和131.6倍(图5)。说明与SCTV01E疫苗相比，SCTV01E-1疫苗针对SARS-CoV-2Omicron BA.2.12.1、BA.2.75和BA.4/5不同的变异株具有更为广谱的中和活性。其中，图5SCTV01E和SCTV01E-1疫苗免疫C57BL/6小鼠后血清抗体效价检测(GeoMean±SD)结果。
综上所述，五组分疫苗针对Omicron新亚型变异株具有广谱的中和能力，因覆盖了更多的变异位点，有希望对多种变异毒株产生交叉保护能力，提高对变异株感染的保护率。
虽然前述已经用说明和实施例的方式对本发明进行了细节描述，但其目的在于理解方便，本领域普通技术人员显然可以对本发明的技术方案作出各种变形和改进，而不会偏离附加的权利要求的精神或范围。
序列列表




















参考文献
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 (11)

  1. 一种提高SARS-CoV-2突变毒株ECD抗原免疫原性/抗原三聚体稳定性的方法，该方法通过
    构建包含SEQ ID No:8所示的氨基酸序列,或其免疫原性片段和/或免疫原性变体的ECD抗原，从而
    ECD为稳定的prefusion构象的三聚体形式；
    优选地，突变毒株为含有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和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之至少任一的高风险突变毒株；
    优选地，该毒株为Omicron(BA.4/BA.5)；
    优选地，该ECD抗原和选自以下的一种或多种佐剂共同施予受试者：
    铝佐剂、油乳佐剂、Toll样受体(TLR)激动剂、免疫增强剂的组合、微生物类佐剂、蜂胶佐剂、左旋咪唑佐剂、脂质体佐剂、中药佐剂及小肽类佐剂；
    优选地，油乳佐剂包含角鲨烯成分；
    Toll样受体(TLR)激动剂包含吸附在铝盐上的CpG或单磷酰脂质A(MPL)；和
    免疫增强剂的组合包含QS-21和/或MPL。
  2. 一种提高SARS-CoV-2突变毒株ECD抗原免疫原性//抗原三聚体稳定性的方法，该方法通过构建编码包含SEQ ID No:8所示的氨基酸序列,或其免疫原性片段和/或免疫原性变体的多核苷酸，从而表达稳定的prefusion构象的三聚体形式ECD；
    优选地，突变毒株为含有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和N969K之至少任一的高风险突变毒株；
    更优选地，该毒株为Omicron(BA.4/BA.5)；
    最优选地，构建包含SEQ ID No:7所示的核苷酸序列或其片段的多核苷酸。
  3. 一种免疫原性/抗原三聚体稳定性提高的SARS-CoV-2突变毒株ECD免疫原性蛋白/肽，其特征在于，该免疫原性蛋白/肽包含SEQ ID No:8所示的氨基酸序列,或其免疫原性片段和/或免疫原性变体，
    该ECD免疫原性蛋白/肽为稳定的prefusion构象的三聚体形式；
    优选地，突变毒株为含有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和N969K之至少任一的高风险突变毒株；
    优选地,该毒株为Omicron(BA.4/BA.5)。
  4. 一种多核苷酸，其编码如权利要求3所述的免疫原性蛋白/肽，
    优选地，包含SEQ ID No:7所示的核苷酸序列。
  5. 一种免疫原性组合物，其特征在于，包含
    如权利要求3所述的免疫原性蛋白/肽，或
    如权利要求4所述的多核苷酸，和
    药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合；
    任选地，包含佐剂。
  6. 权利要求的5免疫原性组合物，其特征在于，进一步包含
    SEQ ID No:16、SEQ ID No:20、SEQ ID No:28或SEQ ID No:32至少之任一所示的氨基酸序列,或其免疫原性片段和/或免疫原性变体，或
    SEQ ID No:15、SEQ ID No:19、SEQ ID No:27或SEQ ID No:31至少之任一所示的核苷酸序列或其活性变体。
  7. 权利要求的5免疫原性组合物，其特征在于，佐剂选自以下的一种或多种：
    铝佐剂、油乳佐剂、Toll样受体(TLR)激动剂、免疫增强剂的组合、微生物类佐剂、蜂胶佐剂、左旋咪唑佐剂、脂质体佐剂、中药佐剂及小肽类佐剂；
    优选地，油乳佐剂包含角鲨烯成分；
    Toll样受体(TLR)激动剂包含吸附在铝盐上的CpG或单磷酰脂质A(MPL)；和
    免疫增强剂的组合包含QS-21和/或MPL。
  8. 权利要求3所述的免疫原性蛋白/肽、权利要求4所述的多核苷酸和权利要求5或6所述的免疫复合物预防或治疗SARS-CoV-2突变毒株引起的疾病的用途，优选地，突变毒株为高风险突变毒株；
    优选地，突变毒株为含有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、和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和N969K之至少任一的高风险突变毒株；
    优选地，该毒株包含D614G突变(B.1)、Beta(B.1.351)、Alpha(B.1.1.7)、Delta(B.1.617.2)、P.1、B.1.427，B.1.429和/或Omicron(BA.1、BA.4/BA.5)；
    更优选地，该毒株包含Alpha(B.1.1.7)、Beta(B.1.351)、Delta(B.1.617.2)和Omicron(BA.1、BA.4/BA.5)。
  9. 权利要求3所述的免疫原性蛋白/肽、权利要求4所述的多核苷酸和权利要求5或6所述的免疫复合物在制备预防或治疗SARS-CoV-2突变毒株引起的疾病的疫苗或药物中的用途，优选地，突变毒株为高风险突变毒株；
    优选地，突变毒株为含有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、和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和N969K之至少任一的高风险突变毒株；
    优选地，该毒株包含D614G突变(B.1)、Beta(B.1.351)、Alpha(B.1.1.7)、Delta(B.1.617.2)P.1、B.1.427，B.1.429和/或Omicron(BA.1、BA.4/BA.5)；
    更优选地，该毒株包含Alpha(B.1.1.7)、Beta(B.1.351)、Delta(B.1.617.2)和/或Omicron(BA.1、BA.4/BA.5)。
  10. 一种改造过的SARS-CoV-2 BA.4/BA.5 ECD序列，其包含
    SEQ ID No:6所示的氨基酸序列或其免疫原性片段和/或免疫原性变体。
  11. 一种编码权利要求10的氨基酸序列或其免疫原性片段和/或免疫原性变体，优选地，其为SEQ ID No:5所示的核苷酸序列或其免疫原性片段和/或免疫原性变体。
PCT/CN2023/111051 2022-08-08 2023-08-03 一种可诱导广谱中和活性重组五组分新冠病毒三聚体蛋白疫苗的制备及应用 WO2024032468A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210946805 2022-08-08
CN202210946805.X 2022-08-08

Publications (1)

Publication Number Publication Date
WO2024032468A1 true WO2024032468A1 (zh) 2024-02-15

Family

ID=89850876

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/111051 WO2024032468A1 (zh) 2022-08-08 2023-08-03 一种可诱导广谱中和活性重组五组分新冠病毒三聚体蛋白疫苗的制备及应用

Country Status (1)

Country Link
WO (1) WO2024032468A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113185613A (zh) * 2021-04-13 2021-07-30 武汉大学 新型冠状病毒s蛋白及其亚单位疫苗
US20210246170A1 (en) * 2020-01-31 2021-08-12 Janssen Pharmaceuticals, Inc. Compositions and Methods for Preventing and Treating Coronavirus Infection - SARS-COV-2 Vaccines
CN113388041A (zh) * 2020-03-12 2021-09-14 厦门大学 具有融合前早期构象的SARS-CoV-2 S三聚体蛋白及其应用
CN114702556A (zh) * 2022-03-22 2022-07-05 中国人民解放军军事科学院军事医学研究院 一种冠状病毒rbd变异体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210246170A1 (en) * 2020-01-31 2021-08-12 Janssen Pharmaceuticals, Inc. Compositions and Methods for Preventing and Treating Coronavirus Infection - SARS-COV-2 Vaccines
CN113388041A (zh) * 2020-03-12 2021-09-14 厦门大学 具有融合前早期构象的SARS-CoV-2 S三聚体蛋白及其应用
CN113185613A (zh) * 2021-04-13 2021-07-30 武汉大学 新型冠状病毒s蛋白及其亚单位疫苗
CN114702556A (zh) * 2022-03-22 2022-07-05 中国人民解放军军事科学院军事医学研究院 一种冠状病毒rbd变异体及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHING-LIN HSIEH ET AL.: "Structure-Based Design of Prefusion-Stabilized SARS-CoV-2 Spikes", SCIENCE, vol. 369, no. 6510, 23 July 2020 (2020-07-23), XP055780339, ISSN: 0036-8075, DOI: 10.1126/science.abd0826 *

Similar Documents

Publication Publication Date Title
Saunders et al. Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses
Van Kampen et al. Safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans
KR20220154121A (ko) 코로나바이러스 면역원성 조성물 및 이의 용도
Sathaliyawala et al. Assembly of human immunodeficiency virus (HIV) antigens on bacteriophage T4: a novel in vitro approach to construct multicomponent HIV vaccines
CN110951756A (zh) 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
Kim et al. Development of spike receptor-binding domain nanoparticles as a vaccine candidate against SARS-CoV-2 infection in ferrets
US9731006B2 (en) Attenuated recombinant vesicular stomatitis virus vaccine vectors comprising modified matrix proteins
Li et al. SARS-CoV-2 vaccine candidates in rapid development
JP2018515141A (ja) 新規のバキュロウイルスベクター及び使用の方法
WO2022148455A1 (en) Identification and uses of peptide sequences of sars-cov-2 t cell and b cell epitopes
Zhang et al. Biochemical and antigenic characterization of the structural proteins and their post-translational modifications in purified SARS-CoV-2 virions of an inactivated vaccine candidate
Cao et al. A single vaccine protects against SARS-CoV-2 and influenza virus in mice
JPH0768267B2 (ja) フラビウイルス抗原
WO2023160654A1 (zh) 一种可诱导广谱中和活性重组多组分新冠病毒三聚体蛋白疫苗的制备及应用
EP3013960A1 (en) Modified matrix proteins of vesicular stomatitis virus
US20170312303A1 (en) Compositions comprising ch848 envelopes and uses thereof
WO2023001259A1 (zh) 一种可诱导广谱中和活性重组多价新冠病毒三聚体蛋白疫苗的制备及应用
Pecetta et al. Immunology and technology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines
WO2024032468A1 (zh) 一种可诱导广谱中和活性重组五组分新冠病毒三聚体蛋白疫苗的制备及应用
US20190231866A1 (en) Methods for safe induction of cross-clade immunity against hiv infection in humans
WO2023111725A1 (en) Sars-cov-2 vaccines
WO2022253134A1 (zh) 一种提高SARS-CoV-2突变毒株ECD抗原免疫原性/抗原三聚体稳定性的方法
CN115477703A (zh) 一种提高SARS-CoV-2突变毒株ECD抗原免疫原性/抗原三聚体稳定性的方法
CN118119646A (zh) 一种可诱导广谱中和活性重组五组分新冠病毒三聚体蛋白疫苗的制备及应用
Zhou et al. One HA stalk topping multiple heads as a novel influenza vaccine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23851689

Country of ref document: EP

Kind code of ref document: A1