WO2022083760A1 - 融合蛋白及其应用 - Google Patents
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- WO2022083760A1 WO2022083760A1 PCT/CN2021/125834 CN2021125834W WO2022083760A1 WO 2022083760 A1 WO2022083760 A1 WO 2022083760A1 CN 2021125834 W CN2021125834 W CN 2021125834W WO 2022083760 A1 WO2022083760 A1 WO 2022083760A1
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Definitions
- the present application relates to the field of biomedicine, in particular to a receptor binding domain (RBD) comprising a SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof and/or a SARS-CoV-2 spike protein (S protein) Protein) fusion proteins of N-terminal domain (NTD) or functionally active fragments thereof, compositions and uses thereof.
- RBD receptor binding domain
- S protein SARS-CoV-2 spike protein
- S protein S protein
- S protein S protein
- S protein S protein
- NTD N-terminal domain
- the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) is considered to be the most important antigenic target region for inducing neutralizing antibodies in the body.
- RBD can focus the neutralizing antibodies stimulated by the body on the receptor binding against the virus, which can improve the immunogenicity and immune efficiency of the vaccine.
- NTD N-terminal domain of the SARS-CoV-2 spike protein
- S protein SARS-CoV-2 spike protein
- the present application provides a receptor binding domain (RBD) comprising the SARS-CoV-2 spike protein (S protein) or its functionally active fragment and/or the N-terminal of the SARS-CoV-2 spike protein (S protein) Fusion proteins, compositions and applications of structural domains (NTDs) or functionally active fragments thereof, which have one or more of the following properties: 1) the fusion proteins are immunogenic; 2) the COVID-19 subtypes comprising the fusion proteins The unit vaccine is more immunogenic and can induce more neutralizing antibodies.
- the application provides a fusion protein comprising: 1) a receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or A plurality of polypeptides selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- RBD receptor binding domain
- S protein S protein
- HBsAg hepatitis B surface antigen
- the RBD is derived from SARS-CoV-2 wild type or a mutant thereof.
- the SARS-CoV-2 mutant is selected from any of the group consisting of Gamma mutants, Beta mutants, Delta mutants, and Alpha mutants.
- the RBD is mutated at one or more amino acid positions selected from the group consisting of K417, L452, T478, E484, and N501 as compared to the RBD of wild-type SAR-CoV-2.
- the RBD comprises one or more of amino acid mutations K417T/N, L452R, T478K, E484K, and N501Y.
- the RBD comprises K417T, E484K, and N501Y amino acid mutations.
- the RBD comprises K417N, E484K and N501Y amino acid mutations.
- the RBD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 18, 19, 76, 83, 97-108.
- the P2 or functionally active fragment thereof comprises an epitope peptide of tetanus toxin.
- the epitope peptide of the tetanus toxin comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64-66.
- the P2 or functionally active fragment thereof is directly or indirectly linked to the N-terminus or C-terminus of the RBD or functionally active fragment thereof.
- the RBD or functionally active fragment thereof is fused in-frame with the P2 or functionally active fragment thereof.
- the foldon domain or functionally active fragment thereof comprises an amino acid residue at the C-terminus of bacteriophage T4 fibrin.
- the foldon domain or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67-69 and 78.
- the foldon domain or functionally active fragment thereof is directly or indirectly linked to the N-terminus or C-terminus of the RBD or functionally active fragment thereof.
- the RBD or functionally active fragment thereof is fused in-frame to the foldon domain or functionally active fragment thereof.
- the ferritin or a functionally active fragment thereof comprises Trichoplusia ferritin or a functionally active fragment thereof.
- the Trichoderma ferritin or functionally active fragment thereof comprises a heavy or light chain of Trichoderma ferritin.
- the heavy chain of Trichoplusia ferritin comprises the amino acid sequence shown in SEQ ID NO:70.
- the light chain of the Trichoplusia ferritin comprises the amino acid sequence shown in SEQ ID NO:71.
- the ferritin or a functionally active fragment thereof comprises Helicobacter pylori ferritin or a functionally active fragment thereof.
- the ferritin or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70-72.
- ferritin or functionally active fragment thereof is linked directly or indirectly to the N-terminus or C-terminus of the RBD or functionally active fragment thereof.
- the RBD or functionally active fragment thereof is fused in-frame with the ferritin or functionally active fragment thereof.
- the hepatitis B surface antigen or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:73.
- the hepatitis B surface antigen or a functionally active fragment thereof is directly or indirectly linked to the N-terminus or C-terminus of the RBD or a functionally active fragment thereof.
- the RBD or functionally active fragment thereof is fused in-frame with the hepatitis B surface antigen or functionally active fragment thereof.
- the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-17, 80 and 86.
- the present application provides a fusion protein comprising: 1) a SARS-CoV-2 spike protein (S protein) N-terminal domain (NTD) or a functionally active fragment thereof; and 2) one or more A polypeptide selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- S protein S protein N-terminal domain
- a polypeptide selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- the NTD is derived from SARS-CoV-2 wild type or a mutant thereof.
- the SARS-CoV-2 mutant is selected from any of the group consisting of Gamma mutants, Beta mutants, Delta mutants, and Alpha mutants.
- the NTD comprises a mutation at one or more amino acid positions selected from the group consisting of L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.
- the NTD comprises one or more amino acid mutations selected from the group consisting of L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.
- the NTD comprises the L18F, D80A, D215G, L242-244del and R246I mutations.
- the NTD comprises L18F, T20N, P26S, D138Y and R246I amino acid mutations.
- the NTD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 37, 38, 77, 84, and 109-111.
- the P2 or functionally active fragment thereof comprises an epitope peptide of tetanus toxin.
- the epitope peptide of the tetanus toxin comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64-66.
- the P2 or functionally active fragment thereof is directly or indirectly linked to the N-terminus or C-terminus of the NTD or functionally active fragment thereof.
- the NTD or functionally active fragment thereof is fused in-frame with the P2 or functionally active fragment thereof.
- the foldon domain or functionally active fragment thereof comprises an amino acid residue at the C-terminus of bacteriophage T4 fibrin.
- the foldon domain or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67-69 and 78.
- the foldon domain or functionally active fragment thereof is directly or indirectly linked to the N-terminus or C-terminus of the NTD or functionally active fragment thereof.
- the NTD or functionally active fragment thereof is fused in-frame to the foldon domain or functionally active fragment thereof.
- the ferritin or a functionally active fragment thereof comprises Trichoplusia ferritin or a functionally active fragment thereof.
- the Trichoderma ferritin or functionally active fragment thereof comprises a heavy or light chain of Trichoderma ferritin.
- the heavy chain of Trichoplusia ferritin comprises the amino acid sequence shown in SEQ ID NO:70.
- the light chain of the Trichoplusia ferritin comprises the amino acid sequence shown in SEQ ID NO:71.
- the ferritin or a functionally active fragment thereof comprises Helicobacter pylori ferritin or a functionally active fragment thereof.
- the ferritin or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70-72.
- the ferritin or functionally active fragment thereof is directly or indirectly linked to the N-terminus or C-terminus of the NTD or functionally active fragment thereof.
- the NTD or functionally active fragment thereof is fused in-frame with the ferritin or functionally active fragment thereof.
- the hepatitis B surface antigen or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:73.
- the hepatitis B surface antigen or a functionally active fragment thereof is directly or indirectly linked to the N-terminus or C-terminus of the NTD or a functionally active fragment thereof.
- the NTD or functionally active fragment thereof is fused in-frame with the hepatitis B surface antigen or functionally active fragment thereof.
- the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 20-36, 81 and 87.
- the present application provides a fusion protein comprising: 1) the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) SARS- CoV-2 spike protein (S protein) N-terminal domain (NTD) or its functionally active fragment.
- RBD receptor binding domain
- S protein SARS-CoV-2 spike protein
- NTD N-terminal domain
- the fusion protein further comprises one or more polypeptides selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, And hepatitis B surface antigen (HBsAg) or its functionally active fragment.
- P2 or a functionally active fragment thereof a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, And hepatitis B surface antigen (HBsAg) or its functionally active fragment.
- HBsAg hepatitis B surface antigen
- the RBD is derived from SARS-CoV-2 wild type or a mutant thereof.
- the SARS-CoV-2 mutant is selected from any of the group consisting of Gamma mutants, Beta mutants, Delta mutants, and Alpha mutants.
- the RBD is mutated at one or more amino acid positions selected from the group consisting of K417, L452, T478, E484, and N501 compared to the RBD of wild-type SAR-CoV-2.
- the RBD comprises one or more of amino acid mutations K417T/N, L452R, T478K, E484K, and N501Y.
- the RBD comprises K417T, E484K, and N501Y amino acid mutations.
- the RBD comprises K417N, E484K and N501Y amino acid mutations.
- the RBD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 18, 19, 76, 83, 97-108.
- the NTD is derived from SARS-CoV-2 wild type or a mutant thereof.
- the SARS-CoV-2 mutant is selected from any of the group consisting of Gamma mutants, Beta mutants, Delta mutants, and Alpha mutants.
- the NTD comprises a mutation at one or more amino acid positions selected from the group consisting of L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.
- the NTD comprises one or more amino acid mutations selected from the group consisting of L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.
- the NTD comprises the L18F, D80A, D215G, L242-244del and R246I mutations.
- the NTD comprises L18F, T20N, P26S, D138Y and R246I amino acid mutations.
- the NTD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 37, 38, 77, 84, and 109-111.
- the RBD or functionally active fragment thereof is directly or indirectly linked to the NTD or functionally active fragment thereof.
- the RBD or functionally active fragment thereof is fused in-frame with the NTD or functionally active fragment thereof.
- the P2 or functionally active fragment thereof comprises an epitope peptide of tetanus toxin.
- the epitope peptide of the tetanus toxin comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64-66.
- the P2 or functionally active fragment thereof is directly or indirectly linked to the RBD or functionally active fragment thereof and/or the NTD or functionally active fragment thereof.
- the P2 or functionally active fragment thereof is fused in-frame with the RBD or functionally active fragment thereof and/or the NTD or functionally active fragment thereof.
- the N-terminus of the P2 or a functionally active fragment thereof is directly or indirectly linked to the C-terminus of the RBD or a functionally active fragment thereof, and the N-terminus of the RBD or a functionally active fragment thereof is connected to the The C-terminus of the NTD or its functionally active fragment is linked directly or indirectly.
- the foldon domain or functionally active fragment thereof comprises an amino acid residue at the C-terminus of bacteriophage T4 fibrin.
- the amino acid residue at the C-terminus of the phage T4 fibrin comprises the amino acid sequence set forth in SEQ ID NO:78.
- the foldon domain or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67-69 and 78.
- the foldon domain or functionally active fragment thereof is directly or indirectly linked to the RBD or functionally active fragment thereof and/or the NTD or functionally active fragment thereof.
- the foldon domain or functionally active fragment thereof is fused in-frame with the RBD or functionally active fragment thereof and/or the NTD or functionally active fragment thereof.
- the N-terminus of the foldon domain or its functionally active fragment is directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment, and the RBD or its functionally active N-terminus is linked to the RBD or its functionally active N-terminus.
- the C-terminus of the NTD or its functionally active fragment is linked directly or indirectly.
- the N-terminus of the foldon domain or a functionally active fragment thereof is directly or indirectly linked to the C-terminus of the P2 or a functionally active fragment thereof, and the N-terminus of the P2 or a functionally active fragment thereof is linked to The C-terminus of the RBD or its functionally active fragment is directly or indirectly connected, and the RBD or its functionally active N-terminus is directly or indirectly connected to the NTD or its functionally active C-terminus.
- the ferritin or a functionally active fragment thereof comprises Trichoplusia ferritin or a functionally active fragment thereof.
- the Trichoderma ferritin or functionally active fragment thereof comprises a heavy or light chain of Trichoderma ferritin.
- the heavy chain of Trichoplusia ferritin comprises the amino acid sequence shown in SEQ ID NO:70.
- the light chain of the Trichoplusia ferritin comprises the amino acid sequence shown in SEQ ID NO:71.
- the ferritin or a functionally active fragment thereof comprises Helicobacter pylori ferritin or a functionally active fragment thereof.
- the ferritin or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70-72.
- ferritin or functionally active fragment thereof is directly or indirectly linked to the RBD or functionally active fragment thereof and/or the NTD or functionally active fragment thereof.
- ferritin or functionally active fragment thereof is fused in-frame with the RBD or functionally active fragment thereof and/or the NTD or functionally active fragment thereof.
- the N-terminus of the ferritin or a functionally active fragment thereof is directly or indirectly linked to the C-terminus of the RBD or a functionally active fragment thereof, and the N-terminus of the RBD or a functionally active fragment thereof is linked to the The C-terminus of the NTD or its functionally active fragment is linked directly or indirectly.
- the hepatitis B surface antigen or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:73.
- the hepatitis B surface antigen or functionally active fragment thereof is directly or indirectly linked to the RBD or functionally active fragment thereof and/or the NTD or functionally active fragment thereof.
- the hepatitis B surface antigen or functionally active fragment thereof is fused in-frame with the RBD or functionally active fragment thereof and/or the NTD or functionally active fragment thereof.
- the N-terminus of the HBsAg or its functionally active fragment is directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment, and the RBD or its functionally active N-terminus is linked to the RBD or its functionally active N-terminus.
- the C-terminus of the NTD or its functionally active fragment is linked directly or indirectly.
- the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 39-44, 79, 85 and 96.
- the present application provides an immunogenic composition comprising the fusion protein.
- the immunogenic composition comprises a first component comprising the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and a second component comprising a fusion protein.
- RBD receptor binding domain
- S protein SARS-CoV-2 spike protein
- the immunogenic composition comprises a first component comprising a fusion protein; and a second component comprising a SARS-CoV-2 stinger
- S protein spike protein
- NTD N-terminal domain
- the immunogenic composition comprises a first component comprising the fusion protein; and a second component comprising the fusion protein fusion protein.
- the immunogenic composition is formulated according to the following weight ratios: 1) the first component (1-15 parts by weight); and/or 2) the second component ( 1-15 parts by weight).
- the immunogenic composition comprises: 1) 5-60 ⁇ g of the first component; and/or 2) 5-60 ⁇ g of the second component.
- the application provides an immunogenic composition
- a first component comprising the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) ) or a functionally active fragment thereof; and a second component comprising the SARS-CoV-2 spike protein (S protein) N-terminal domain (NTD) or a functionally active fragment thereof.
- RBD receptor binding domain
- NTD N-terminal domain
- the RBD is derived from SARS-CoV-2 wild type or a mutant thereof.
- the SARS-CoV-2 mutant is selected from any of the group consisting of Gamma mutants, Beta mutants, Delta mutants, and Alpha mutants.
- the RBD is mutated at one or more amino acid positions selected from the group consisting of K417, L452, T478, E484, and N501 compared to the RBD of wild-type SAR-CoV-2.
- the RBD comprises one or more of amino acid mutations K417T/N, L452R, T478K, E484K, and N501Y.
- the RBD comprises K417T, E484K and N501Y amino acid mutations.
- the RBD comprises K417N, E484K and N501Y amino acid mutations.
- the RBD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 18, 19, 76, 83, 97-108.
- the RBD is derived from SARS-CoV-2 wild type or a mutant thereof.
- the SARS-CoV-2 mutant is selected from any of the group consisting of Gamma mutants, Beta mutants, Delta mutants, and Alpha mutants.
- the NTD comprises a mutation at one or more amino acid positions selected from the group consisting of L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.
- the NTD comprises one or more amino acid mutations selected from the group consisting of L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.
- the NTD comprises the L18F, D80A, D215G, L242-244del and R246I mutations.
- the NTD comprises L18F, T20N, P26S, D138Y and R246I amino acid mutations.
- the NTD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 37, 38, 77, 84, and 109-111.
- the immunogenic composition is formulated according to the following weight ratios: 1) the first component (1-15 parts by weight); and/or 2) the second component ( 1-15 parts by weight).
- the immunogenic composition comprises: 1) 5-60 ⁇ g of the first component; and/or 2) 5-60 ⁇ g of the second component.
- the RBD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 18-19, 76, 83, 97-108.
- the NTD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 37-38, 77, 84, and 109-111.
- the present application provides a pharmaceutical composition comprising the fusion protein, or the immunogenic composition, and optionally a pharmaceutically acceptable excipient.
- the present application provides the use of the fusion protein or the immunogenic composition in preparing a vaccine.
- the vaccine is used to prevent and/or treat COVID-19.
- the present application provides the fusion protein or the immunogenic composition for treating and/or preventing COVID-19.
- the application provides a method for preparing a COVID-19 subunit vaccine, comprising:
- the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies, comprising:
- the present application provides a method for treating and/or preventing COVID-19, comprising administering to a subject the fusion protein or the immunogenic composition or the COVID-19 subtype unit vaccine.
- Figure 1 shows the results of RBD-P2-6*HIS protein verification by SDS-PAGE electrophoresis and WB.
- Figure 2 shows the results of verifying NTD-P2-6*HIS (GP101150-8) protein by SDS-PAGE electrophoresis and WB.
- Figure 3 shows the results of the verification of NTD-RBD-foldon-8*HIS protein by SDS-PAGE electrophoresis and WB.
- Figure 4 shows the amino acid sequence alignment of the sequencing results of NTD-RBD-foldon-6 ⁇ His (Gamma mutant).
- Figure 5 shows the size exclusion chromatographic profile of NTD-RBD-foldon (Gamma mutant) protein.
- Figure 6 shows the results of SDS-PAGE electrophoresis and Western blot verification of NTD-RBD-foldon (Gamma mutant) protein.
- Figure 7 shows the amino acid sequence alignment of the sequencing results of NTD-RBD-foldon-6 ⁇ His (Beta mutant strain).
- Figure 8 shows the size exclusion chromatogram of NTD-RBD-foldon (Beta mutant) protein.
- Figure 9 shows the results of SDS-PAGE electrophoresis and Western blot verification of NTD-RBD-foldon (Beta mutant) protein.
- fusion protein generally refers to a protein molecule with biological functional activity obtained by genetic engineering technology.
- the fusion protein can be a fusion protein composed of RBD or its functionally active fragment and any of the following sequences: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment , Hepatitis B surface antigen HBsAg or its functionally active fragment.
- the fusion protein can be a fusion protein composed of NTD or its functionally active fragment and any of the following sequences: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, hepatitis B Surface antigen HBsAg or its functionally active fragment.
- the fusion protein can be a fusion protein composed of NTD or its functionally active fragment and RBD or its functionally active fragment.
- the fusion protein can be a fusion protein composed of NTD or its functionally active fragment, RBD or its functionally active fragment and any of the following fragments: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, hepatitis B surface antigen HBsAg or its functionally active fragment.
- GISAID uses the full genome sequence (EPI_ISL_402124) of the strain hCoV-19/WIV04/2019 (WIV04) as the official reference sequence.
- the WIV04 strain is generally defined as the wild or original strain.
- SARS-CoV-2 wild type generally refers to the WIV04 strain.
- P2 generally refers to the epitope peptide of tetanus toxin.
- P2 can be a peptide fragment of epitope peptides 830 to 844 of tetanus toxin or a mutant thereof.
- P2 may be a peptide segment of epitope peptides 830 to 845 of tetanus toxin or a mutant thereof.
- P2 can be a peptide fragment of epitope peptides 829 to 844 of tetanus toxin or a mutant thereof.
- P2 may be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 829 to 844 of tetanus toxin.
- P2 can be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptide 830 to 845 of tetanus toxin.
- P2 can be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 830 to 844 of tetanus toxin.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the term "foldon domain” generally refers to residues at the C-terminus of phage T4 fibrin.
- the foldon domain may be the 27 residues or mutants of the C-terminus of bacteriophage T4 fibrin.
- the foldon domain may be obtained by truncating 27 residues at the C-terminus of phage T4 fibrin or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminus or C-terminus. Truncated or grown.
- “mutant” generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the terms “ferritin”, “ferritin” generally refer to Trichoplusia ferritin or Helicobacter pylori ferritin.
- Trichoderma ferritin has heavy and light chains (ferritin LC and ferritin HC).
- H. pylori ferritin has a single-chain structure.
- the ferritin may be a mutant of Trichoplusia or Helicobacter pylori ferritin.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- LC light chain of Trichoplusia ferritin
- HC Trichoplusia ferritin heavy chain
- hepatitis B surface antigen generally refers to a coat protein in the outermost envelope of the hepatitis B virus.
- the amino acid sequence of hepatitis B surface antigen can be the sequence corresponding to the protein sequence accession number AAA45524, ANJ76941, CAA24234 or AAC34729 in NCBI, or the N-terminal or C-terminal 1, 2, 3, 4, 5 can be appropriately truncated or added. , or 6 or 10 amino acids, or a mutation of the protein, such as deletion, substitution or insertion of one or more amino acids.
- the term "functionally active fragment” generally refers to a fragment having similar biological activities to RBD, NTD, P2, foldon domain, ferritin, HBsAg.
- the term "immunogenic composition” generally refers to a subunit composition.
- a subunit composition is a combination in which the components have been isolated and purified to at least 50%, at least 60%, 70%, 80%, 90% purity prior to combining the components to form the antigenic composition thing.
- the subunit composition can be an aqueous solution of a water-soluble protein.
- the subunit composition may comprise a detergent.
- the subunit composition may comprise a non-ionic, zwitterionic or ionic detergent.
- the subunit composition can include lipids.
- the immunogenic composition can include RBD or functionally active fragment thereof and NTD or functionally active fragment thereof.
- the immunogenic composition can include RBD or a functionally active fragment thereof and a fusion protein comprising NTD or a functionally active fragment thereof.
- an immunogenic composition can include a fusion protein comprising RBD or a functionally active fragment thereof and an NTD or functionally active fragment thereof.
- the immunogenic composition can include fusion proteins comprising RBD or functionally active fragments thereof and fusion proteins comprising NTD or functionally active fragments thereof.
- the immunogenic composition may also include an adjuvant.
- the adjuvant may include an aluminum salt (eg, aluminum hydroxide gel (alum) or aluminum phosphate), but may also be a calcium, iron, or zinc salt, or may be acylated tyrosine or acylated Insoluble suspensions of sugars, cationically or anionically derivatized polysaccharides or polyphosphazenes.
- the immunogenic composition may also be selected to be a preferential inducer of a Th1-type response.
- a preferential inducer of a Th1-type response may include monophosphoryl lipid A or a derivative thereof.
- the adjuvant can be a combination of monophosphoryl lipid A (eg, 3-de-O-acylated monophosphoryl lipid A (3D-MPL)) and an aluminum salt.
- An adjuvant-enhancing system may include a combination of monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in WO94/00153, or quenching of QS21 with cholesterol as disclosed in WO96/33739 A composition that makes the reactogenicity less reactive.
- the adjuvant may also be the adjuvant described in WO95/17210 containing QS21, 3D-MPL and tocopherol in an oil-in-water emulsion.
- the adjuvant may be a uniform droplet emulsion formed by microfluidization under high pressure after mixing Tween 80, sorbitan trioleate and squalene.
- the adjuvant may comprise unmethylated CpG of the oligonucleotide (WO 96/02555).
- the term "parts by weight” generally refers to the weight ratio of the first component and the second component of the immunogenic composition.
- the immunogenic composition may contain the following components per dose: 5-60 ⁇ g of the first component and 5-60 ⁇ g of the second component.
- the immunogenic composition may comprise 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 30 ⁇ g or 40 ⁇ g of the first component.
- the immunogenic composition may comprise 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 30 ⁇ g or 40 ⁇ g of the second component.
- the term "RBD” refers to the receptor binding domain of the SARS-CoV-2 spike protein (S protein).
- the RBD can be a peptide segment between amino acids 310-560 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, or a truncated N-terminal or C-terminal 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids.
- the RBD can be a peptide segment between amino acids 319-541 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, and the N-terminal or C-terminal can also be appropriately truncated 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids.
- the RBD can be a peptide segment between amino acids 331-524 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, or the N-terminal or C-terminal 1, 2 can be appropriately truncated , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids.
- “mutant” generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the RBD may comprise the RBD of SARS-CoV-2 (Gamma mutant).
- the RBD can comprise the RBD of SARS-CoV-2 (Beta mutant).
- the RBD can comprise the RBD of SARS-CoV-2 (WIV04-1).
- the term "NTD" refers to the N-terminal domain of the SARS-CoV-2 spike protein (S protein).
- the NTD can be a peptide segment between amino acids 13-353 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, or the N-terminus or C-terminus can be appropriately truncated 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids.
- the NTD can be a peptide segment between amino acids 13-303 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof.
- the NTD can be a peptide segment between amino acids 14-304 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof. In the present application, the NTD can be a peptide segment between amino acids 18-353 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the NTD may comprise the NTD of SARS-CoV-2 (Gamma mutant). In certain embodiments, the NTD may comprise the NTD of SARS-CoV-2 (Beta mutant). In certain embodiments, the NTD may comprise the NTD of SARS-CoV-2 (WIV04-1).
- S protein also referred to as “Spike protein” or “Spike protein” generally refers to the capsid surface glycoprotein of coronaviruses.
- SARS-COV-2 binds to the ACE2 receptor through the S protein and invades cells.
- the S protein is composed of 1213 amino acids and contains a transmembrane region, including a fragment of at least 1213 amino acids from the N-terminus or from the 14th amino acid of the capsid surface glycoprotein from the coronavirus, or the corresponding region from other SARS viruses.
- S1 protein is subunit 1 of S protein, mainly refers to the fragment from the N-terminal or the 14th amino acid to the 685th amino acid.
- the mutant may comprise any one or several mutant strains (mutants) of SAR-CoV-2 currently known.
- the Gamma mutant strain can refer to the SARS-CoV-2 surface glycoprotein amino acid sequence of the B.1.1.28 lineage with the GenBank accession number QRN46961.1.
- the Gamma mutant strain can refer to the SARS-CoV-2 surface glycoprotein amino acid sequence of the B.1.1.28 lineage with GenBank accession number QLF80256.1.
- the Gamma mutant strain can refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of the P.1 lineage with GenBank accession number QVE55289.1.
- the Gamma mutant strain can be the Gamma mutant strain 501Y.V3, and the amino acid sequence of its surface glycoprotein can be shown in SEQ ID NO:88.
- the Beta mutant strain can refer to the SARS-CoV-2 surface glycoprotein amino acid sequence of the B.1.351 lineage with the GenBank accession number QUA12570.1.
- the Beta mutant strain can refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of the B.1 lineage with the GenBank accession number QIZ15537.1.
- the Beta mutant strain can refer to the SARS-CoV-2 surface glycoprotein amino acid sequence of the B.1.351 lineage with the GenBank accession number QVI03430.1.
- the Beta mutant can be a 501Y.V2Beta mutant whose surface glycoprotein amino acid sequence is shown in SEQ ID NO:82.
- hemagglutinin/hemagglutinin protein/HA generally refers to antibodies or other substances that can agglutinate red blood cells, which are found on the surfaces of influenza viruses, measles viruses (as well as many other bacteria and viruses), etc. Found, can attach to the red blood cells of different animals, and cause red blood cells to agglutinate, which can be fatal in severe cases. In this context it refers to the surface glycoprotein hemagglutinin (HA) of influenza virus. Every year, the WHO official website will publish the prediction of the influenza sequence of the year.
- This article refers to the influenza sequence of the northern hemisphere in 2021-2022, that is, the influenza virus type A (H1N1) EPI1661231
- the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
- the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
- the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
- the carrier also includes a carrier having a variety of the above-mentioned functions.
- the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
- the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
- the term "host cell” generally refers to an individual cell, cell line that can or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or an individual cell, cell line capable of expressing an antibody or antigen-binding fragment thereof described herein or cell cultures.
- the host cells can include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the fusion proteins described herein.
- the host cells can be obtained by transfecting cells in vitro using the vectors described herein.
- the host cell can be a prokaryotic cell (eg E.
- the host cell is a mammalian cell.
- the mammalian cells can be CHO cells.
- the application provides a fusion protein comprising: 1) a receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or A plurality of polypeptides selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- RBD receptor binding domain
- S protein S protein
- HBsAg hepatitis B surface antigen
- the RBD or its functionally active fragment can be derived from the RBD of the SARS-CoV-2 wild-type S protein or the RBD of the SARS-CoV-2 mutant S protein.
- the RBD or functionally active fragment thereof may comprise amino acid mutations at one or more amino acid positions in K417, L452, T478, E484, and N501.
- the RBD or its functionally active fragment may comprise the RBD of the Gamma mutant S protein.
- the RBD or functionally active fragment thereof may comprise one or more amino acid mutations in K417T, L452R, T478K, E484K and N501Y.
- the RBD can comprise the amino acid sequence set forth in SEQ ID NO:83.
- the RBD or its functionally active fragment may comprise the RBD of the Beta mutant S protein.
- the RBD or functionally active fragment thereof may comprise one or more amino acid mutations in K417N, E484K and N501Y.
- the RBD can comprise the amino acid sequence set forth in SEQ ID NO:76.
- the P2 or its functionally active fragment may comprise an epitope peptide of tetanus toxin.
- P2 can be a peptide fragment of epitope peptides 830 to 844 of tetanus toxin or a mutant thereof.
- P2 may be a peptide segment of epitope peptides 830 to 845 of tetanus toxin or a mutant thereof.
- P2 can be a peptide fragment of epitope peptides 829 to 844 of tetanus toxin or a mutant thereof.
- P2 may be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 829 to 844 of tetanus toxin.
- P2 can be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptide 830 to 845 of tetanus toxin.
- P2 can be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 830 to 844 of tetanus toxin.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NOs: 64-66.
- the P2 or its functionally active fragment may be directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.
- the P2 or its functionally active fragment may be directly or indirectly linked to the N-terminus of the RBD or its functionally active fragment.
- the P2 or its functionally active fragment may be directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment.
- the RBD or a functionally active fragment thereof may be fused in-frame with the P2 or a functionally active fragment thereof.
- the foldon domain or its functionally active fragment may comprise an amino acid residue at the C-terminus of phage T4 fibrin.
- the foldon domain or a functionally active fragment thereof may comprise 27 amino acid residues or mutants of the C-terminal of phage T4 fibrin.
- the foldon domain may be a 27 residue truncation of the C-terminus of phage T4 fibrin or a truncation obtained by adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N- or C-terminus.
- short body or growth body generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the foldon domain or functionally active fragment thereof may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 67-69 and 78.
- the foldon domain or its functionally active fragment may be directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.
- the foldon domain or its functionally active fragment may be directly or indirectly linked to the N-terminus of the RBD or its functionally active fragment.
- the foldon domain or its functionally active fragment may be directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment.
- the RBD or a functionally active fragment thereof may be fused in-frame with the foldon domain or a functionally active fragment thereof.
- the ferritin or a functionally active fragment thereof may comprise Trichoplusia ferritin or a functionally active fragment thereof.
- the Trichoderma ferritin or a functionally active fragment thereof may comprise a heavy chain or a light chain of Trichoderma ferritin.
- the ferritin may be a mutant of Trichoplusia or Helicobacter pylori ferritin.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- light chain of Trichoplusia ferritin can be used interchangeably with “ferritin LC”.
- Trichoplusia ferritin heavy chain can be used interchangeably with “ferritin HC”.
- H. pylori ferritin is used interchangeably with "HP ferritin”.
- the heavy chain of Trichoplusia ferritin may comprise the amino acid sequence shown in SEQ ID NO:70.
- the light chain of Trichoplusia ferritin may comprise the amino acid sequence shown in SEQ ID NO:71.
- the ferritin or a functionally active fragment thereof may comprise Helicobacter pylori ferritin or a functionally active fragment thereof.
- the Helicobacter pylori ferritin or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO:72.
- ferritin or its functionally active fragment may comprise the amino acid sequence shown in any one of SEQ ID NOs: 70-72.
- ferritin or its functionally active fragment may be directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.
- ferritin or a functionally active fragment thereof may be linked directly or indirectly to the N-terminus of the RBD or a functionally active fragment thereof.
- ferritin or a functionally active fragment thereof may be linked directly or indirectly to the C-terminus of the RBD or a functionally active fragment thereof.
- the RBD or a functionally active fragment thereof may be fused in-frame with the ferritin or a functionally active fragment thereof.
- hepatitis B surface antigen generally refers to a coat protein in the outermost envelope of the hepatitis B virus.
- the amino acid sequence of hepatitis B surface antigen can be the sequence corresponding to the protein sequence accession number AAA45524, ANJ76941, CAA24234 or AAC34729 in NCBI, or the N-terminal or C-terminal 1, 2, 3, 4, 5 can be appropriately truncated or added. , or 6 or 10 amino acids, or a mutation of the protein, such as deletion, substitution or insertion of one or more amino acids.
- hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:73.
- the hepatitis B surface antigen (HBsAg) or its functionally active fragment may be directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.
- hepatitis B surface antigen (HBsAg) or its functionally active fragment may be directly or indirectly linked to the N-terminus of the RBD or its functionally active fragment.
- hepatitis B surface antigen (HBsAg) or its functionally active fragment may be directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment.
- the RBD or a functionally active fragment thereof may be fused in-frame with the hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- HBsAg hepatitis B surface antigen
- the "directly or indirectly connected” usually refers to two different connection modes: direct connection and indirect connection of two sequences, wherein the direct connection refers to the connection between the two sequences without any artificial addition.
- the remaining sequences such as flexible linker, rigid linker or shearable linker, etc. are involved.
- Indirect connection means that the two sequences are connected by artificially adding connecting sequences, such as flexible Linker, rigid Linker or cleavable Linker.
- the flexible Linker can comprise the amino acid sequence of GSGSG (SEQ ID NO:89).
- the linker sequence may comprise the amino acid sequence of SFTVEKGIYQTSNF (SEQ ID NO:90).
- the fusion protein may also comprise a signal peptide.
- the signal peptide sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 91) can be added to the N-segment of the fusion protein.
- the fusion protein can comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-17, 80 and 86.
- the present application provides a fusion protein comprising: 1) a SARS-CoV-2 spike protein (S protein) N-terminal domain (NTD) or a functionally active fragment thereof; and 2) one or more A polypeptide selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- S protein S protein N-terminal domain
- a polypeptide selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- the NTD or its functionally active fragment can be derived from the NTD of the SARS-CoV-2 wild-type S protein, or can be derived from the NTD of the SARS-CoV-2 mutant S protein.
- the NTD or functionally active fragment thereof may comprise amino acids at one or more amino acid positions of L18, T19, T20, P26, D80, D138, R190, D215, L242-244 and R246 mutation.
- the NTD or functionally active fragment thereof may comprise one or more amino acid mutations in L18F, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.
- the NTD or its functionally active fragment may comprise the NTD of the Gamma mutant S protein.
- the NTD may comprise L18F, T20N, P26S, D138Y and R190S amino acid mutations.
- the NTD may comprise the amino acid sequence set forth in SEQ ID NO:84.
- the NTD or its functionally active fragment may comprise, for example, the NTD of the Beta mutant S protein.
- the NTD or functionally active fragment thereof may comprise the L18F, D80A, D215G, L242-244del and R246I mutations.
- the NTD may comprise the amino acid sequence set forth in SEQ ID NO:77.
- the P2 or its functionally active fragment may comprise an epitope peptide of tetanus toxin.
- P2 can be a peptide fragment of epitope peptides 830 to 844 of tetanus toxin or a mutant thereof.
- P2 may be a peptide segment of epitope peptides 830 to 845 of tetanus toxin or a mutant thereof.
- P2 can be a peptide fragment of epitope peptides 829 to 844 of tetanus toxin or a mutant thereof.
- P2 may be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 829 to 844 of tetanus toxin.
- P2 can be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 830 to 845 of tetanus toxin.
- P2 may be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 830 to 844 of tetanus toxin.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NOs: 64-66.
- the P2 or its functionally active fragment may be directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.
- the P2 or its functionally active fragment may be directly or indirectly linked to the N-terminus of the NTD or its functionally active fragment.
- the P2 or its functionally active fragment may be directly or indirectly linked to the C-terminus of the NTD or its functionally active fragment.
- the NTD or a functionally active fragment thereof may be fused in-frame with the P2 or a functionally active fragment thereof.
- the foldon domain or a functionally active fragment thereof may comprise an amino acid residue at the C-terminus of phage T4 fibrin.
- the amino acid residue at the C-terminus of the phage T4 fibrin may comprise the amino acid sequence shown in SEQ ID NO:78.
- the foldon domain or a functionally active fragment thereof may comprise the 27 amino acid residues or mutants of the C-terminus of phage T4 fibrin.
- the foldon domain may be a 27 residue truncation of the C-terminus of phage T4 fibrin or a truncation obtained by adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N- or C-terminus.
- short body or growth body generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the foldon domain or functionally active fragment thereof may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 67-69 and 78.
- the foldon domain or its functionally active fragment may be directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.
- the foldon domain or its functionally active fragment may be directly or indirectly linked to the N-terminus of the NTD or its functionally active fragment.
- the foldon domain or its functionally active fragment may be directly or indirectly linked to the C-terminus of the NTD or its functionally active fragment.
- the NTD or a functionally active fragment thereof may be fused in-frame with the foldon domain or a functionally active fragment thereof.
- the ferritin or a functionally active fragment thereof may comprise Trichoplusia ferritin or a functionally active fragment thereof.
- the Trichoderma ferritin or a functionally active fragment thereof may comprise a heavy chain or a light chain of Trichoderma ferritin.
- the ferritin may be a mutant of Trichoplusia or Helicobacter pylori ferritin.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- light chain of Trichoplusia ferritin can be used interchangeably with “ferritin LC”.
- Trichoplusia ferritin heavy chain can be used interchangeably with “ferritin HC”.
- H. pylori ferritin is used interchangeably with "HP ferritin”.
- the heavy chain of Trichoplusia ferritin may comprise the amino acid sequence shown in SEQ ID NO:70.
- the light chain of Trichoplusia ferritin may comprise the amino acid sequence shown in SEQ ID NO:71.
- the ferritin or a functionally active fragment thereof may comprise Helicobacter pylori ferritin or a functionally active fragment thereof.
- the Helicobacter pylori ferritin or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO:72.
- ferritin or its functionally active fragment may comprise the amino acid sequence shown in any one of SEQ ID NOs: 70-72.
- ferritin or its functionally active fragment may be directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.
- ferritin or a functionally active fragment thereof may be linked directly or indirectly to the N-terminus of the NTD or a functionally active fragment thereof.
- ferritin or a functionally active fragment thereof may be linked directly or indirectly to the C-terminus of the NTD or a functionally active fragment thereof.
- the NTD or a functionally active fragment thereof may be fused in-frame with the ferritin or a functionally active fragment thereof.
- hepatitis B surface antigen generally refers to a coat protein in the outermost envelope of the hepatitis B virus.
- the amino acid sequence of hepatitis B surface antigen can be the sequence corresponding to the protein sequence accession number AAA45524, ANJ76941, CAA24234 or AAC34729 in NCBI, or the N-terminal or C-terminal 1, 2, 3, 4, 5 can be appropriately truncated or added. , or 6 or 10 amino acids, or a mutation of the protein, such as deletion, substitution or insertion of one or more amino acids.
- hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:73.
- the hepatitis B surface antigen (HBsAg) or its functionally active fragment may be directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.
- the hepatitis B surface antigen (HBsAg) or its functionally active fragment may be directly or indirectly linked to the N-terminus of the NTD or its functionally active fragment.
- hepatitis B surface antigen (HBsAg) or its functionally active fragment may be directly or indirectly linked to the C-terminus of the NTD or its functionally active fragment.
- the NTD or a functionally active fragment thereof may be fused in-frame with the hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- HBsAg hepatitis B surface antigen
- the "directly or indirectly connected” usually refers to two different connection modes: direct connection and indirect connection of two sequences, wherein the direct connection refers to the connection between the two sequences without any artificial addition.
- the remaining sequences such as flexible linker, rigid linker or shearable linker, etc. are involved.
- Indirect connection means that the two sequences are connected by artificially adding connecting sequences, such as flexible Linker, rigid Linker or cleavable Linker.
- the flexible Linker can comprise the amino acid sequence of GSGSG (SEQ ID NO:89).
- the linker sequence may comprise the amino acid sequence of SFTVEKGIYQTSNF (SEQ ID NO:90).
- the fusion protein may also comprise a signal peptide.
- the signal peptide sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 91) can be added to the N-segment of the fusion protein.
- the fusion protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 20-36, 81 and 87.
- the present application provides a fusion protein comprising: 1) the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) SARS- CoV-2 spike protein (S protein) N-terminal domain (NTD) or its functionally active fragment.
- RBD receptor binding domain
- S protein SARS-CoV-2 spike protein
- NTD N-terminal domain
- the RBD or its functionally active fragment can be derived from the RBD of the SARS-CoV-2 wild-type S protein or the RBD of the SARS-CoV-2 mutant S protein.
- the RBD or functionally active fragment thereof may comprise amino acid mutations at one or more amino acid positions in K417, L452, T478, E484, and N501.
- the RBD or its functionally active fragment may comprise the RBD of the Gamma mutant S protein.
- the RBD or functionally active fragment thereof may comprise one or more amino acid mutations in K417T, E484K and N501Y.
- the RBD can comprise the amino acid sequence set forth in SEQ ID NO:83.
- the RBD or its functionally active fragment may comprise the RBD of the Beta mutant S protein.
- the RBD or functionally active fragment thereof may comprise one or more amino acid mutations in K417N, E484K and N501Y.
- the RBD can comprise the amino acid sequence set forth in SEQ ID NO:76.
- the NTD or its functionally active fragment can be derived from the NTD of the SARS-CoV-2 wild-type S protein, or can be derived from the NTD of the SARS-CoV-2 mutant S protein.
- the NTD or functionally active fragment thereof may comprise amino acids at one or more amino acid positions of L18, T19, T20, P26, D80, D138, R190, D215, L242-244 and R246 mutation.
- the NTD or functionally active fragment thereof may comprise one or more amino acid mutations in L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.
- the NTD or its functionally active fragment may comprise the NTD of the Gamma mutant S protein.
- the NTD may comprise L18F, T20N, P26S, D138Y and R190S amino acid mutations.
- the NTD may comprise the amino acid sequence set forth in SEQ ID NO:84.
- the NTD or its functionally active fragment may comprise the NTD of the S protein of the Beta mutant.
- the NTD or functionally active fragment thereof may comprise the L18F, D80A, D215G, L242-244del and R246I mutations.
- the NTD may comprise the amino acid sequence set forth in SEQ ID NO:77.
- the RBD or a functionally active fragment thereof is directly or indirectly linked to the NTD or a functionally active fragment thereof.
- the N-terminus of the RBD or its functionally active fragment is directly or indirectly linked to the C-terminus of the NTD or its functionally active fragment.
- the C-terminus of the RBD or its functionally active fragment is directly or indirectly linked to the N-terminus of the NTD or its functionally active fragment.
- the RBD or a functionally active fragment thereof is fused in-frame with the NTD or a functionally active fragment thereof.
- the fusion protein may also include one or more polypeptides selected from the group consisting of P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and Hepatitis B surface antigen (HBsAg) or its functionally active fragment.
- the P2 or its functionally active fragment may comprise an epitope peptide of tetanus toxin.
- P2 can be a peptide fragment of epitope peptides 830 to 844 of tetanus toxin or a mutant thereof.
- P2 may be a peptide segment of epitope peptides 830 to 845 of tetanus toxin or a mutant thereof.
- P2 can be a peptide fragment of epitope peptides 829 to 844 of tetanus toxin or a mutant thereof.
- P2 may be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 829 to 844 of tetanus toxin.
- P2 can be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptide 830 to 845 of tetanus toxin.
- P2 can be a peptide fragment obtained by truncating or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N-terminal or C-terminal of the epitope peptides 830 to 844 of tetanus toxin.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the epitope peptide of the tetanus toxin may comprise the amino acid sequence shown in any one of SEQ ID NOs: 64-66.
- the P2 or a functionally active fragment thereof may be directly or indirectly linked to the RBD or a functionally active fragment thereof and/or the NTD or a functionally active fragment thereof.
- the N-terminus of the RBD or a functionally active fragment thereof may be directly or indirectly linked to the C-terminus of the NTD or a functionally active fragment thereof, and the N-terminus of the P2 or a functionally active fragment thereof may be linked to the RBD or The C-terminus of its functionally active fragment is linked directly or indirectly.
- the P2 or a functionally active fragment thereof may be fused in-frame with the RBD or a functionally active fragment thereof and the NTD or a functionally active fragment thereof.
- the foldon domain or a functionally active fragment thereof may comprise an amino acid residue at the C-terminus of phage T4 fibrin.
- the amino acid residue at the C-terminus of the phage T4 fibrin comprises the amino acid sequence set forth in SEQ ID NO:78.
- the foldon domain or a functionally active fragment thereof may comprise the 27 amino acid residues or mutants of the C-terminus of phage T4 fibrin.
- the foldon domain may be a 27 residue truncation of the C-terminus of phage T4 fibrin or a truncation obtained by adding 1, 2, 3, 4, 5, or 6 or 10 amino acids to the N- or C-terminus.
- short body or growth body generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- the foldon domain or functionally active fragment thereof may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 67-69 and 78.
- the foldon domain or a functionally active fragment thereof is directly or indirectly linked to the RBD or a functionally active fragment thereof and/or the NTD or a functionally active fragment thereof.
- the N-terminus of the foldon domain or its functionally active fragment may be directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment, and the RBD or its functionally active N-terminus may be linked to the NTD or The C-terminus of its functionally active fragment is linked directly or indirectly.
- the foldon domain or a functionally active fragment thereof is fused in-frame with the RBD or a functionally active fragment thereof and the NTD or a functionally active fragment thereof.
- the N-terminus of the foldon domain or its functionally active fragment is directly or indirectly connected to the C-terminus of the P2 or its functionally active fragment, and the N-terminus of the P2 or its functionally active fragment is connected to the The C-terminus of the RBD or its functionally active fragment is directly or indirectly linked, and the RBD or its functionally active N-terminus is directly or indirectly linked to the NTD or its functionally active C-terminus.
- the P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, the RBD or a functionally active fragment thereof, and the NTD or a functionally active fragment thereof are fused in-frame.
- the ferritin or a functionally active fragment thereof comprises Trichoplusia ferritin or a functionally active fragment thereof.
- the Trichoderma ferritin or its functionally active fragment includes the heavy chain or the light chain of Trichoderma ferritin.
- the ferritin may be a mutant of Trichoplusia or Helicobacter pylori ferritin.
- mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
- light chain of Trichoplusia ferritin can be used interchangeably with “ferritin LC”.
- Trichoplusia ferritin heavy chain can be used interchangeably with “ferritin HC”.
- H. pylori ferritin is used interchangeably with "HP ferritin”.
- the heavy chain of Trichoplusia ferritin may comprise the amino acid sequence shown in SEQ ID NO:70.
- the light chain of Trichoplusia ferritin may comprise the amino acid sequence shown in SEQ ID NO:71.
- the ferritin or a functionally active fragment thereof may comprise Helicobacter pylori ferritin or a functionally active fragment thereof.
- ferritin or its functionally active fragment may comprise the amino acid sequence shown in any one of SEQ ID NOs: 70-72.
- ferritin or a functionally active fragment thereof may be directly or indirectly linked to the RBD or a functionally active fragment thereof and/or the NTD or a functionally active fragment thereof.
- the N-terminus of the ferritin or its functionally active fragment may be directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment, and the RBD or its functionally active N-terminus may be linked to the NTD or its functionally active N-terminus.
- the C-termini of the functionally active fragments are linked directly or indirectly.
- the N-terminus of the Helicobacter pylori ferritin or its functionally active fragment may be directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment, and the RBD or its functionally active N-terminus may be linked to the The C-terminus of the NTD or its functionally active fragment is linked directly or indirectly.
- the H. pylori ferritin or a functionally active fragment thereof may be fused in-frame with the RBD or a functionally active fragment thereof and the NTD or a functionally active fragment thereof.
- hepatitis B surface antigen generally refers to a coat protein in the outermost envelope of the hepatitis B virus.
- the amino acid sequence of hepatitis B surface antigen can be the sequence corresponding to the protein sequence accession number AAA45524, ANJ76941, CAA24234 or AAC34729 in NCBI, or the N-terminal or C-terminal 1, 2, 3, 4, 5 can be appropriately truncated or added. , or 6 or 10 amino acids, or a mutation of the protein, such as deletion, substitution or insertion of one or more amino acids.
- the hepatitis B surface antigen or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO:73.
- the hepatitis B surface antigen or a functionally active fragment thereof may be directly or indirectly linked to the RBD or a functionally active fragment thereof and/or the NTD or a functionally active fragment thereof.
- the N-terminus of the hepatitis B surface antigen or its functionally active fragment may be directly or indirectly connected to the C-terminus of the RBD or its functionally active fragment, and the RBD or its functionally active N-terminus may be connected to the RBD or its functionally active N-terminus.
- the C-terminus of the NTD or its functionally active fragment is linked directly or indirectly.
- the hepatitis B surface antigen or a functionally active fragment thereof is fused in-frame with the RBD or a functionally active fragment thereof and the NTD or a functionally active fragment thereof.
- the "directly or indirectly connected” usually refers to two different connection modes: direct connection and indirect connection of two sequences, wherein the direct connection refers to the connection between the two sequences without any artificial addition.
- the remaining sequences such as flexible linker, rigid linker or shearable linker, etc. are involved.
- Indirect connection means that the two sequences are connected by artificially adding connecting sequences, such as flexible Linker, rigid Linker or cleavable Linker.
- the flexible Linker can comprise the amino acid sequence of GSGSG (SEQ ID NO:89).
- the linker sequence may comprise the amino acid sequence of SFTVEKGIYQTSNF (SEQ ID NO:90).
- the fusion protein may also comprise a signal peptide.
- the signal peptide sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 91) can be added to the N-segment of the fusion protein.
- the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 39-44, 79, 85 and 96.
- the present application provides an immunogenic composition comprising the fusion protein.
- the term "immunogenic composition” generally refers to a subunit composition.
- a subunit composition is a combination in which the components have been isolated and purified to at least 50%, at least 60%, 70%, 80%, 90% purity prior to combining the components to form the antigenic composition thing.
- the subunit composition can be an aqueous solution of a water-soluble protein.
- the subunit composition may comprise a detergent.
- the subunit composition may comprise a non-ionic, zwitterionic or ionic detergent.
- the subunit composition can include lipids.
- the immunogenic composition can include RBD or functionally active fragment thereof and NTD or functionally active fragment thereof.
- the immunogenic composition can include RBD or a functionally active fragment thereof and a fusion protein comprising NTD or a functionally active fragment thereof.
- an immunogenic composition can include a fusion protein comprising RBD or a functionally active fragment thereof and an NTD or functionally active fragment thereof.
- the immunogenic composition can include fusion proteins comprising RBD or functionally active fragments thereof and fusion proteins comprising NTD or functionally active fragments thereof.
- the immunogenic composition may also include one or more human influenza virus hemagglutinin proteins, HA.
- the HA may comprise the amino acid sequence of any one of SEQ ID NOs: 92-95.
- influenza strain selected from the influenza hemagglutinin is a circulating strain published on the WHO official website every year, for example, the circulating strain published on the WHO official website in 2021 refers to type A ( H1N1)EPI1661231
- the preparation of a vaccine composition for preventing influenza and new coronavirus includes 50 ⁇ g of the above-mentioned various types of HA protein and 50 ⁇ g of mutated NTD-RBD-foldon protein and adjuvants commonly used in vaccine combinations, such as AS03 adjuvant.
- the immunogenic composition may comprise a first component and a second component
- the first component may comprise a fusion protein of an RBD or a functionally active fragment thereof and an NTD or a functionally active fragment thereof as described herein
- the fusion protein can also comprise one or more polypeptides selected from the group consisting of: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen (HBsAg ) or a functionally active fragment thereof
- the second component may comprise the RBD or a functionally active fragment thereof, the NTD or a functionally active fragment thereof, or optionally one or several human influenza virus hemagglutinin proteins HA .
- the RBD or its functionally active fragment can be derived from the RBD of the SARS-CoV-2 wild-type S protein or the RBD of the SARS-CoV-2 mutant S protein.
- the RBD or functionally active fragment thereof may comprise amino acid mutations at one or more amino acid positions in K417, L452, T478, E484, and N501.
- the RBD or its functionally active fragment may comprise the RBD of the Gamma mutant S protein.
- the RBD or functionally active fragment thereof may comprise one or more amino acid mutations in K417T, E484K and N501Y.
- the RBD can comprise the amino acid sequence set forth in SEQ ID NO:83.
- the RBD or its functionally active fragment may comprise the RBD of the Beta mutant S protein.
- the RBD or functionally active fragment thereof may comprise one or more amino acid mutations in K417N, E484K and N501Y.
- the RBD may comprise the amino acid sequence set forth in SEQ ID NO:76.
- the NTD or its functionally active fragment can be derived from the NTD of the SARS-CoV-2 wild-type S protein, or can be derived from the NTD of the SARS-CoV-2 mutant S protein.
- the NTD or functionally active fragment thereof may comprise amino acids at one or more amino acid positions of L18, T19, T20, P26, D80, D138, R190, D215, L242-244 and R246 mutation.
- the NTD or functionally active fragment thereof may comprise one or more amino acid mutations in L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.
- the NTD or its functionally active fragment may comprise the NTD of the Gamma mutant S protein.
- the NTD may comprise L18F, T20N, P26S, D138Y and R190S amino acid mutations.
- the NTD may comprise the amino acid sequence set forth in SEQ ID NO:84.
- the NTD or its functionally active fragment may comprise the NTD of the S protein of the Beta mutant.
- the NTD or functionally active fragment thereof may comprise the L18F, D80A, D215G, L242-244del and R246I mutations.
- the NTD may comprise the amino acid sequence set forth in SEQ ID NO:77.
- the immunogenic composition may also include an adjuvant.
- the adjuvant may include an aluminum salt (eg, aluminum hydroxide gel (alum) or aluminum phosphate), but may also be a calcium, iron, or zinc salt, or may be acylated tyrosine or acylated Insoluble suspensions of sugars, cationically or anionically derivatized polysaccharides or polyphosphazenes.
- the immunogenic composition may also be selected to be a preferential inducer of a Th1-type response.
- a preferential inducer of a Th1-type response may include monophosphoryl lipid A or a derivative thereof.
- the adjuvant can be a combination of monophosphoryl lipid A (eg, 3-de-O-acylated monophosphoryl lipid A (3D-MPL)) and an aluminum salt.
- An adjuvant-enhancing system may include a combination of monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in WO94/00153, or quenching of QS21 with cholesterol as disclosed in WO96/33739 A composition that makes the reactogenicity less reactive.
- the adjuvant may also be the adjuvant described in WO95/17210, which contains QS21, 3D-MPL and tocopherol in an oil-in-water emulsion.
- the adjuvant may be a uniform droplet emulsion formed by microfluidization under high pressure after mixing Tween 80, sorbitan trioleate and squalene.
- the adjuvant may comprise unmethylated CpG of the oligonucleotide (WO 96/02555).
- the immunogenic composition comprises a first component comprising the fusion protein; and a second component comprising the fusion protein .
- the immunogenic composition can be formulated according to the following weight ratios: 1) the first component (1-15 parts by weight); and/or 2) the second component (1) -15 parts by weight).
- the immunogenic composition may comprise: 1) 5-60 ⁇ g of the first component; and/or 2) 5-60 ⁇ g of the second component.
- the term "parts by weight” generally refers to the weight ratio of the first component and the second component of the immunogenic composition.
- the immunogenic composition may contain the following components per dose: 5-60 ⁇ g of the first component and 5-60 ⁇ g of the second component.
- the immunogenic composition may comprise 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 30 ⁇ g or 40 ⁇ g of the first component.
- the immunogenic composition may comprise 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 30 ⁇ g or 40 ⁇ g of the second component.
- the application provides an immunogenic composition
- a first component comprising the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) ) or a functionally active fragment thereof; and a second component comprising the SARS-CoV-2 spike protein (S protein) N-terminal domain (NTD) or a functionally active fragment thereof.
- RBD receptor binding domain
- NTD N-terminal domain
- the immunogenic composition can be formulated according to the following weight ratios: 1) the first component (1-15 parts by weight); and/or 2) the second component (1) -15 parts by weight).
- the immunogenic composition may comprise: 1) 5-60 ⁇ g of the first component; and/or 2) 5-60 ⁇ g of the second component.
- the RBD may comprise the amino acid sequence shown in any one of SEQ ID NOs: 18-19.
- the NTD comprises the amino acid sequence shown in any one of SEQ ID NOs: 37-38.
- the term "parts by weight” generally refers to the weight ratio of the first component and the second component of the immunogenic composition.
- the immunogenic composition may contain the following components per dose: 5-60 ⁇ g of the first component and 5-60 ⁇ g of the second component.
- the immunogenic composition may comprise 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 30 ⁇ g or 40 ⁇ g of the first component.
- the immunogenic composition may comprise 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 30 ⁇ g or 40 ⁇ g of the second component.
- the immunogenic composition may comprise the first component NTD-RBD-foldon protein 20-100 ⁇ g; and the second component NTD-foldon protein 20-100 ⁇ g, or RBD-foldon protein 20-100 ⁇ g, Or influenza virus hemagglutinin protein 15 ⁇ 80 ⁇ g.
- the immunogenic composition comprises 20-100 ⁇ g of NTD-RBD-foldon (Gamma mutant) protein, 20-100 ⁇ g of NTD-foldon (Gamma mutant) protein, and a lyoprotectant
- the lyoprotectants were prepared according to 1% (w/v) sucrose; 2% (w/v) glycine; 0.02% (w/v) Tween 80; and 10 mM PB buffer (Na 2 ) at pH 7.5 HPO 4 , NaH 2 PO 4 ).
- the immunogenic composition comprises NTD-RBD-foldon (Gamma mutant) protein 20-100 ⁇ g, RBD-foldon (Gamma mutant) protein 20-100 ⁇ g, and a lyoprotectant
- the lyoprotectants were prepared according to 1% (w/v) sucrose; 2% (w/v) glycine; 0.02% (w/v) Tween 80; and 10 mM PB buffer (Na 2 ) at pH 7.5 HPO 4 , NaH 2 PO 4 ).
- the immunogenic composition comprises 20-100 ⁇ g of NTD-RBD-foldon (Gamma mutant) protein, 15-180 ⁇ g of influenza virus hemagglutinin protein, and a lyoprotectant, the described
- the lyoprotectants were 1% (w/v) sucrose; 2% (w/v) glycine; 0.02% (w/v) Tween 80; and 10 mM PB buffer (Na 2 HPO 4 , pH 7.5) NaH 2 PO 4 ) formulation.
- the present invention provides a vaccine comprising the fusion protein, or the immunogenic composition, and optionally a pharmaceutically acceptable adjuvant.
- the vaccine comprises 0.5 mL of the fusion protein or immunogenic composition, and 0.5 mL, optionally, of a pharmaceutically acceptable adjuvant.
- the adjuvant of the present invention is selected from one of squalene, MF59, AS03, monophosphatidyl lipid A, flagellin, CpG-ODN, muramyl dipeptide, and small molecules of aluminum or calcium salt or variety. These adjuvants are well known in the art and are available through several commercial sources.
- the adjuvant is an aluminum hydroxide adjuvant.
- the adjuvant is MF59 adjuvant.
- the adjuvant is preferably an AS03 adjuvant.
- the components of the AS03 adjuvant include 10.69 mg of squalene, 11.86 mg of alpha-tocopherol, 4.86 mg of polysorbate 80, 3.53 mg of sodium chloride, 0.09 mg of potassium chloride, and 0.51 mg of hydrogen phosphate Disodium, 0.09 mg of potassium dihydrogen phosphate.
- composition of the MF59 adjuvant includes 4.5% squalene, 0.5% Tween 80, 0.5% Span85.
- the vaccine comprises NTD-RBD-foldon protein 20-100 ⁇ g, 1% (w/v) sucrose, 2% (w/v) glycine, 0.02% (w/v) Tween 80 and 10 mM PB buffer ( Na2HPO4 , NaH2PO4 ) , pH 7.5, and AS03 adjuvant.
- the vaccine comprises 20-100 ⁇ g of NTD-RBD-foldon protein, 15-45 ⁇ g of HA of H1N1 strain, 15-45 ⁇ g of HA of H3N2 strain, and 15-45 ⁇ g of HA of B/Washington/02/2019 strain HA 15-45 ⁇ g, 1% (w/v) sucrose, 2% (w/v) glycine, 0.02% (w/v) Tween 80 and 10 mM PB buffer pH 7.5 (Na 2 HPO 4 , NaH 2 PO 4 ), and AS03 adjuvant.
- the vaccine comprises NTD-RBD-foldon protein 20-100 ⁇ g, H1N1 strain HA 15-45 ⁇ g, H3N2 strain HA 15-45 ⁇ g, B/Washington/02/2019 strain HA 15-45 ⁇ g, and HA 15-45 ⁇ g of B/PHUKET/3073/2013 strain, 1% (w/v) sucrose, 2% (w/v) glycine, 0.02% (w/v) Tween 80 and pH 7 .5 in 10 mM PB buffer ( Na2HPO4 , NaH2PO4 ) , and AS03 adjuvant.
- the vaccine comprises 20-100 ⁇ g of NTD-RBD-foldon protein, 15-45 ⁇ g of HA of H1N1 strain, 15-45 ⁇ g of HA of H3N2 strain, and 15-45 ⁇ g of HA of B/Washington/02/2019 strain HA 15-45 ⁇ g, 1% (w/v) sucrose, 2% (w/v) glycine, 0.02% (w/v) Tween 80 and 10 mM PB buffer pH 7.5 (Na 2 HPO 4 , NaH 2 PO 4 ), and MF59 adjuvant.
- the vaccine comprises NTD-RBD-foldon protein 20-100 ⁇ g, H1N1 strain HA 15-45 ⁇ g, H3N2 strain HA 15-45 ⁇ g, B/Washington/02/2019 strain HA 15-45 ⁇ g, and HA 15-45 ⁇ g of B/PHUKET/3073/2013 strain, 1% (w/v) sucrose, 2% (w/v) glycine, 0.02% (w/v) Tween 80 and pH 7 .5 in 10 mM PB buffer ( Na2HPO4 , NaH2PO4 ) , and MF59 adjuvant.
- the vaccine comprises 50 ⁇ g of NTD-RBD-foldon protein, 45 ⁇ g of HA of H1N1 strain, 45 ⁇ g of HA of H3N2 strain, 45 ⁇ g of HA of B/Washington/02/2019 strain, and B HA 45 ⁇ g of /PHUKET/3073/2013 strain, 1% (w/v) sucrose, 2% (w/v) glycine, 0.02% (w/v) Tween 80 and 10 mM PB buffer pH 7.5 ( Na2HPO4 , NaH2PO4 ) , and AS03 adjuvant.
- the vaccine comprises 50 ⁇ g of NTD-RBD-foldon protein, 45 ⁇ g of HA of H1N1 strain, 45 ⁇ g of HA of H3N2 strain, 45 ⁇ g of HA of B/Washington/02/2019 strain, and B HA 45 ⁇ g of /PHUKET/3073/2013 strain, 1% (w/v) sucrose, 2% (w/v) glycine, 0.02% (w/v) Tween 80 and 10 mM PB buffer pH 7.5 ( Na2HPO4 , NaH2PO4 ) , and MF59 adjuvant.
- the RBD-NTD-foldon (Beta mutant) may comprise the amino acid sequence shown in SEQ ID NO:79.
- the RBD-NTD-foldon (Gamma mutant) may comprise the amino acid sequence shown in SEQ ID NO:85.
- the RBD-NTD-foldon (WIV04-1) may comprise the amino acid sequence shown in SEQ ID NO:96.
- the immunogenic composition may also comprise one or several human influenza virus hemagglutinin proteins, HA.
- the HA may comprise the amino acid sequence of any one of SEQ ID NOs: 92-95.
- the RBD may also comprise the amino acid sequence shown in any one of SEQ ID NOs: 97-108.
- the NTD may also comprise the amino acid sequence shown in any one of SEQ ID NOs: 109-111.
- the application also provides one or more isolated nucleic acid molecules that encode the fusion proteins described herein.
- the nucleic acid molecule may comprise mRNA.
- the present application provides a pharmaceutical composition comprising the fusion protein, or the immunogenic composition, and optionally a pharmaceutically acceptable excipient.
- the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or nonionic surfactants agent, etc.
- the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration Administration or via subcutaneous depot.
- compositions can be used to inhibit or delay the development or progression of a disease or disorder, and/or can reduce and/or stabilize the state of a disease or disorder.
- the pharmaceutical composition described in this application may comprise a prophylactically and/or therapeutically effective amount of the fusion protein, or the immunogenic composition.
- the prophylactically and/or therapeutically effective amount is that amount required to prevent and/or treat (at least in part) a disease or disorder and/or any complications thereof in a subject having or at risk of developing it.
- the term "effective amount” generally refers to an amount of a drug that can alleviate or eliminate a disease or symptom in a subject, or prevent or prevent the occurrence of a disease or symptom prophylactically. Generally, this can be determined according to the subject's body weight, age, sex, diet, excretion rate, past medical history, current treatment, time of administration, dosage form, method of administration, route of administration, drug combination, the subject's health
- the specific effective amount will be determined by the condition and potential for cross-infection, allergies, hypersensitivity and side effects, and/or extent of epithelial (or endothelial) tissue disease progression, among others.
- One skilled in the art eg, a physician or veterinarian can proportionally lower or increase the dose according to these or other conditions or requirements.
- the subject may include a human or a non-human animal.
- the non-human animal may be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice and rats.
- non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals.
- the person may be Caucasian, African, Asian, Semitic, or other race, or a hybrid of various races.
- the human can be an elderly, adult, adolescent, child, or infant.
- the effective amount in humans can be extrapolated from the effective amount in experimental animals.
- Freireich et al. describe the correlation of doses (based on milligrams per square meter of body surface) in animals and humans (Freiheim et al., Cancer Chemother. Rep. 50, 219 (1966)).
- Body surface area can be approximately determined from the patient's height and weight. See, eg, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
- the present application provides the use of the fusion protein or the immunogenic composition in preparing a vaccine.
- the vaccine can be used to prevent and/or treat COVID-19.
- the present application provides the fusion protein or the immunogenic composition for treating and/or preventing COVID-19.
- the application provides a method for preparing a COVID-19 subunit vaccine, comprising:
- the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies, comprising:
- the present application provides a method for treating and/or preventing COVID-19, comprising administering to a subject the fusion protein or the immunogenic composition or the COVID-19 subtype unit vaccine.
- a fusion protein comprising: 1) a receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or more selected from the group consisting of Polypeptide: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen (HBsAg) or its functionally active fragment.
- RBD receptor binding domain
- S protein S protein
- HBsAg hepatitis B surface antigen
- SAR-CoV-2 mutant is selected from any one of the group consisting of Gamma mutants, Beta mutants, Delta mutants and Alpha mutants.
- RBD has a mutation at one or more amino acid sites selected from the group compared to the RBD of wild-type SARS-CoV-2 : K417, L452, T478, E484 and N501.
- RBD comprises one or more of amino acid mutations K417T/N, L452R, T478K, E484K, and N501Y.
- fusion protein of any one of embodiments 1-13, wherein the foldon domain or functionally active fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 67-69 and 78.
- ferritin or a functionally active fragment thereof comprises Trichoplusia ferritin or a functionally active fragment thereof.
- Trichoplusia ferritin or functionally active fragment thereof comprises a heavy chain or a light chain of Trichoplusia ferritin.
- fusion protein of any one of embodiments 1-21, wherein the ferritin or a functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70-72.
- fusion protein of any one of embodiments 1-24, wherein the hepatitis B surface antigen or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:73.
- linker may comprise a rigid linker, a flexible linker, or other sequences.
- fusion protein of any one of embodiments 28-29, wherein the linker may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 89-90.
- fusion protein of any one of embodiments 1-30 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-17, 80, and 86.
- a fusion protein comprising: 1) a SARS-CoV-2 spike protein (S protein) N-terminal domain (NTD) or a functionally active fragment thereof; and 2) one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.
- S protein S protein N-terminal domain
- HBsAg hepatitis B surface antigen
- the fusion protein of embodiment 33, wherein the SARS-CoV-2 mutant is selected from any one of the group consisting of a Gamma mutant, a Beta mutant, a Delta mutant, and an Alpha mutant.
- NTD comprises one or more amino acid mutations selected from the group consisting of L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G , L242-244del and R246I.
- NTD comprises L18F, D80A, D215G, L242-244del, and R190S amino acid mutations.
- NTD comprises L18F, T20N, P26S, D138Y, and R246I amino acid mutations.
- fusion protein of any one of embodiments 32-39, wherein the NTD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 37, 38, 77, 84, and 109-111.
- fusion protein of any one of embodiments 32-44, wherein the foldon domain or functionally active fragment thereof comprises an amino acid residue at the C-terminus of bacteriophage T4 fibrin.
- fusion protein of any one of embodiments 32-45, wherein the foldon domain or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67-69 and 78.
- the fusion protein of any one of embodiments 32-48, wherein the ferritin or a functionally active fragment thereof comprises Trichoplusia ferritin or a functionally active fragment thereof.
- Trichoplusia ferritin or a functionally active fragment thereof comprises a heavy chain or a light chain of Trichoderma ferritin.
- the fusion protein of embodiment 50, wherein the heavy chain of Trichoplusia ferritin comprises the amino acid sequence shown in SEQ ID NO:70.
- the fusion protein of any one of embodiments 32-52, wherein the ferritin or a functionally active fragment thereof comprises Helicobacter pylori ferritin or a functionally active fragment thereof.
- the fusion protein of any one of embodiments 32-53, wherein the ferritin or a functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70-72.
- fusion protein of any one of embodiments 32-56, wherein the hepatitis B surface antigen or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:73.
- fusion protein of any one of embodiments 43-59, wherein the direct or indirect linkage may comprise linkage through a linker.
- linker may comprise a rigid linker, a flexible linker or other sequence.
- fusion protein of any one of embodiments 60-61, wherein the linker may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 89-90.
- fusion protein of any one of embodiments 32-62 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 20-36, 81, and 87.
- a fusion protein comprising: 1) a receptor binding domain (RBD) of a SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) a SARS-CoV-2 spike protein (S protein); protein) N-terminal domain (NTD) or a functionally active fragment thereof.
- RBD receptor binding domain
- S protein SARS-CoV-2 spike protein
- S protein S protein
- NTD N-terminal domain
- the fusion protein of embodiment 65, wherein the SARS-CoV-2 mutant is selected from any of the group consisting of a Gamma mutant, a Beta mutant, a Delta mutant, and an Alpha mutant.
- fusion protein of any one of embodiments 64-70, wherein the RBD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 18, 19, 76, 83, 97-108.
- the fusion protein of embodiment 72, wherein the SARS-CoV-2 mutant is selected from any of the group consisting of a Gamma mutant, a Beta mutant, a Delta mutant, and an Alpha mutant.
- NTD comprises one or more amino acid mutations selected from the group consisting of L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del and R246I.
- NTD comprises the L18F, D80A, D215G, L242-244del, and R246I mutations.
- fusion protein of any one of embodiments 64-76, wherein the NTD comprises L18F, D80A, D215G, L242-244del, and R190S amino acid mutations.
- NTD comprises L18F, T20N, P26S, D138Y, and R246I amino acid mutations.
- fusion protein of any one of embodiments 64-78, wherein the NTD comprises the amino acid sequence set forth in any one of SEQ ID NOs: 37, 38, 77, 84, and 109-111.
- fusion protein of any one of embodiments 64-79 further comprising one or more polypeptides selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or its functionally active fragments, and hepatitis B surface antigen (HBsAg) or its functionally active fragments.
- polypeptides selected from the group consisting of P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or its functionally active fragments, and hepatitis B surface antigen (HBsAg) or its functionally active fragments.
- the fusion protein of embodiment 83, wherein the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NOs: 64-66.
- fusion protein of any one of embodiments 64-88, wherein the foldon domain or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67-69 and 78.
- fusion protein according to any one of embodiments 64-89, wherein the foldon domain or its functionally active fragment is directly or directly associated with the RBD or its functionally active fragment and/or the NTD or its functionally active fragment. connected indirectly.
- fusion protein according to any one of embodiments 64-90, wherein the foldon domain or a functionally active fragment thereof is in frame with the RBD or a functionally active fragment thereof and/or the NTD or a functionally active fragment thereof. internal fusion.
- fusion protein according to any one of embodiments 64-92, wherein the N-terminus of the foldon domain or a functionally active fragment thereof is directly or indirectly connected to the C-terminus of the P2 or a functionally active fragment thereof, and The N-terminus of the P2 or its functionally active fragment is directly or indirectly connected to the C-terminus of the RBD or its functionally active fragment, and the N-terminus of the RBD or its functionally active fragment is directly or indirectly connected to the C-terminus of the NTD or its functionally active fragment. or indirectly connected.
- Trichoplusia ferritin or functionally active fragment thereof comprises a heavy chain or a light chain of Trichoplusia ferritin.
- the fusion protein of embodiment 95, wherein the heavy chain of Trichoplusia ferritin comprises the amino acid sequence shown in SEQ ID NO:70.
- ferritin or a functionally active fragment thereof comprises Helicobacter pylori ferritin or a functionally active fragment thereof.
- fusion protein of any one of embodiments 64-98, wherein the ferritin or a functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70-72.
- fusion protein according to any one of embodiments 64-99, wherein the ferritin or its functionally active fragment and the RBD or its functionally active fragment and/or the NTD or its functionally active fragment directly or indirectly connected to the ground.
- fusion protein of any one of embodiments 64-102, wherein the hepatitis B surface antigen or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:73.
- fusion protein according to any one of embodiments 64-104, wherein the hepatitis B surface antigen or its functionally active fragment is in frame with the RBD or its functionally active fragment and/or the NTD or its functionally active fragment. internal fusion.
- fusion protein of any one of embodiments 81-105, wherein the direct or indirect linkage can comprise linkage through a linker.
- the fusion protein of embodiment 106, wherein the linker may comprise a rigid linker, a flexible linker or other sequence.
- the fusion protein of embodiments 81-107, wherein the linker can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 89-90.
- fusion protein of any one of embodiments 64-108 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 39-44, 79, 85, and 96.
- An immunogenic composition comprising the fusion protein of any one of embodiments 1-109.
- the immunogenic composition of embodiment 110 comprising a first component comprising the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and a second component comprising the fusion protein of any one of Embodiments 1-109.
- RBD receptor binding domain
- S protein SARS-CoV-2 spike protein
- the immunogenic composition of embodiment 110 comprising a first component comprising the fusion protein of any one of embodiments 1-109; and a second component,
- the second component comprises the SARS-CoV-2 spike protein (S protein) N-terminal domain (NTD) or a functionally active fragment thereof.
- the immunogenic composition of embodiment 110 comprising a first component comprising the fusion protein of any one of embodiments 1-109; and a second component,
- the second component comprises the fusion protein of any one of Embodiments 1-109.
- the immunogenic composition of any one of embodiments 110-113 formulated in the following weight ratios: 1) the first component (1-15 parts by weight); and/or 2) the first component Two components (1-15 parts by weight).
- immunogenic composition of any one of embodiments 110-114 comprising: 1) 5-60 ⁇ g of the first component; and/or 2) 5-60 ⁇ g of the second component.
- An immunogenic composition comprising a first component comprising the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and The second component, the second component comprises the SARS-CoV-2 spike protein (S protein) N-terminal domain (NTD) or a functionally active fragment thereof.
- RBD receptor binding domain
- NTD N-terminal domain
- the immunogenic composition of embodiment 117, wherein the SARS-CoV-2 mutant is selected from any of the group consisting of: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant .
- the immunogenic composition of embodiment 124, wherein the SARS-CoV-2 mutant is selected from any of the group consisting of: a Gamma mutant, a Beta mutant, a Delta mutant, and an Alpha mutant .
- immunogenic composition of any one of embodiments 116-125, wherein the NTD comprises a mutation at one or more amino acid positions selected from the group consisting of L18, T20, P26, D80, D138, R190, D215, L242-244 and R246.
- the immunogenic composition of any one of embodiments 116-127, wherein the NTD comprises L18F, D80A, D215G, L242-244del, and R246I mutations.
- the immunogenic composition of any one of embodiments 116-131 formulated in the following weight ratios: 1) the first component (1-15 parts by weight); and/or 2) the first component Two components (1-15 parts by weight).
- immunogenic composition of any one of embodiments 111-132 comprising: 1) 5-60 ⁇ g of the first component; and/or 2) 5-60 ⁇ g of the second component.
- nucleic acid molecule of claim 136 comprising mRNA.
- a pharmaceutical composition comprising the fusion protein of any one of embodiments 1-109, or the immunogenic composition of any one of embodiments 110-135, or any one of embodiments 136-137 The nucleic acid molecule of Item, and optionally a pharmaceutically acceptable excipient.
- embodiment 140 The use of embodiment 139, wherein the vaccine is for preventing and/or treating COVID-19.
- fusion protein of any one of embodiments 1-109, or the immunogenic composition of any one of embodiments 110-135, or the nucleic acid molecule of any one of embodiments 136-137, For the treatment and/or prevention of COVID-19.
- a method of preparing a COVID-19 subunit vaccine comprising:
- a method of detecting neutralizing antibodies to SARS-CoV-2 comprising:
- a method of treating and/or preventing COVID-19 comprising administering to a subject the fusion protein of any one of embodiments 1-109, or the immunogen of any one of embodiments 110-135 A sexual composition, or the nucleic acid molecule of any one of Embodiments 136-137, or the COVID-19 subunit vaccine of Embodiment 142.
- Signal peptide addition MGVPAVPEASSPRWGTLLLAIFLAASRGLVAA (SEQ ID NO: 74).
- Vector selection pcDNA3.1(+), codon-optimized according to host CHO cells.
- RBD-P2-6*HIS The primers of RBD-P2-6*HIS were amplified by PCR to obtain fragment PCR products, which were recombined into the target vector pcdna3.1(+) (BamHI-XhoI restriction vector) by multi-segment recombination to obtain RBD-P2- 6*HIS (GP101150-7) recombinant expression plasmid.
- the reaction system is shown in Table 1.
- the above ligation solution was connected in a constant temperature environment of 52°C for 30 min to obtain a recombinant expression plasmid of RBD-P2-6*HIS (GP101150-7).
- Transformation method (1) Pipet 1-3 ⁇ l of the RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid at a concentration of 100 ng/ ⁇ l into 100 ⁇ l of competent cells, gently shake and rotate to mix, Place on ice for 3 minutes. (2) Do not shake the water bath at 42°C for 90s. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 ⁇ l of pre-warmed LB medium at 37°C to each tube, and shake gently at 37°C at 200 rpm for 40 minutes.
- Verification of RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid: (1) Prepare agar plates containing corresponding resistance. (2) Take 100 ⁇ l of bacterial liquid, then spread it on an agar plate containing the corresponding resistance, gently spread the bacteria on the surface of the plate with a sterile glass spreader, and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours to form colonies. (4) Pick the bacteria from the plate, shake the bacteria at 37°C at 250 rpm for 14 hours, use the bacterial solution for PCR identification, and send the positive cloned bacterial solution for sequencing.
- Identification method of cloned plasmid PCR amplification of RBD-P2-6*HIS fragment, identification primer sequence is synthesized by the company's internal primers The expected amplified fragment length is 861bp, PCR reaction adopts 20 ⁇ L system: primer 0.5 ⁇ L, template bacterial solution 2 ⁇ L , polymerase buffer 0.5 ⁇ L, buffer 3 ⁇ L, ddH 2 O 14 ⁇ L. Cycling parameters: pre-denaturation at 96 °C for 3 min; 95 °C for 15 s, 58 °C for 15 s, 72 °C for 20 s, 23 cycles, and a final extension at 72 °C for 1 min.
- the positive clones were screened by bacterial liquid PCR method, and the obtained positive bacterial liquid was shaken at 37°C to extract plasmids and sequenced.
- the correct plasmids were sequenced and compared, and double digested with BamHI--XhoI to obtain two fragments of 861bp and 5372bp.
- CHO cells EXPICHO from Thermo, Inc.
- Day -1 Cell expansion, expand cultured cells to 3 x 10 6 -4 x 10 6 cells/mL, and allow cells to grow overnight.
- Day 0 Transfect cells, measure viable cell density and viability, the cell density should reach 7 ⁇ 10 6 –10 ⁇ 10 6 cells/mL, and the viability reaches 95–99% for transfection, fresh cells expressing culture base, preheated to 37°C, cells were diluted to a final density of 6 ⁇ 10 6 cells/mL, cultured at 37°C at 90 rpm in a 50mm amplitude incubator, 8% CO 2 .
- OPti-PRO SFM medium to prepare transfection reagent and RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid complex (4°C), for example: 1ml CHO cells prepare 40 ⁇ l OPti-PRO SFM add 0.8ug RBD- P2-6*HIS (GP101150-7) recombinant expression plasmids were mixed and placed for 5 minutes; 40 ⁇ l of OPti-PRO SFM was prepared, 3 ⁇ l of Expi Fectamine CHO reagent was added, and 3 ⁇ l of Expi Fectamine CHO reagent was added, mixed for 5 minutes, 1–5 minutes at room temperature, and then the solution was slowly transferred to a shaker flask Shake, shaking the bottle gently during addition.
- the cells were cultured at 37° C., 8% CO 2 in a 50 mm amplitude incubator at 90 rpm. 18-22 hours after transfection, the Enhancer and ExpiCHO Feed were added and the standard protocol was performed.
- B. Electrophoresis: Mix RBD-P2-6*HIS purified protein with loading buffer (RBD-P2-6*HIS purified protein: 5x loading buffer 4:1), boil for 5 min, and perform sampling. First of all 100V constant voltage electrophoresis, you can see that the Marker gradually becomes a thin line. After the marker enters the separation gel, adjust the voltage to 300V and continue electrophoresis until the blue bromophenol blue band reaches the bottom of the gel (about 25min).
- NTD-P2-6*HIS were amplified by PCR to obtain fragment PCR products, which were recombined into the target vector pcdna3.1(+) (BamHI-XhoI digestion vector) by multi-segment recombination to obtain NTD-P2- 6*HIS (GP101150-8) recombinant expression plasmid.
- the reaction system is shown in Table 3.
- Transformation method Pipette 1-3 ⁇ l of the NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid with a concentration of about 100 ng/ ⁇ l into the competent cells of about 100 ⁇ l, gently shake and rotate to mix, ice Set aside for 3 minutes. Do not shake in a 42°C water bath for 90s; place in an ice bath for about 3 minutes; add 500-800 ⁇ l of 37°C pre-warmed LB medium to each tube, and shake gently at 37°C at 200 rpm for 40 minutes.
- Verification of the recombinant expression plasmid of NTD-P2-6*HIS (GP101150-8): (1) Prepare an agar plate containing the corresponding resistance. (2) Take 100 ⁇ l of bacterial liquid, then spread it on an agar plate containing the corresponding resistance, gently spread the bacteria on the surface of the plate with a sterile glass spreader, and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours to form colonies. (4) Pick the bacteria from the plate, shake the bacteria at 37°C at 250 rpm for 14 hours, use the bacterial solution for PCR identification, and send the positive cloned bacterial solution for sequencing.
- Identification method of cloned plasmid PCR amplification of NTD-P2-6*HIS fragment, identification primer sequence is synthesized by the company's internal primers The expected amplified fragment length is 1005bp, PCR reaction adopts 20 ⁇ L system: primer 0.5 ⁇ L, template bacterial solution 2 ⁇ L , polymerase buffer 0.5 ⁇ L, buffer 3 ⁇ L, ddH2O 14 ⁇ L. Cycling parameters: pre-denaturation at 96 °C for 3 min; 95 °C for 15 s, 58 °C for 15 s, 72 °C for 20 s, 23 cycles, and a final extension at 72 °C for 1 min.
- the positive clones were screened by bacterial liquid PCR method, and the obtained positive bacterial liquid was shaken at 37°C to extract plasmids and sequenced.
- the correct plasmids were sequenced and compared, and double digested with BamHI--XhoI to obtain two fragments of 1005bp and 5372bp.
- NTD-P2-6*HIS GP101150-8 recombinant expression plasmid was extracted.
- 1% E. coli cells (stbl3) containing the recombinant expression plasmid of NTD-P2-6*HIS (GP101150-8) were added to 2 ml of LB medium and cultured overnight at 37°C with shaking. After cells were treated, 100 ug of plasmid was extracted using a plasmid extraction kit. The correct plasmids were sequenced and compared, and double digested with BamHI--XhoI to obtain two fragments of 1005bp and 5372bp.
- Day 0 Transfect cells, measure viable cell density and viability, the cell density should reach 7 ⁇ 10 6 –10 ⁇ 10 6 cells/mL, and the viability reaches 95–99% for transfection, fresh cells expressing culture base, preheated to 37°C, cells were diluted to a final density of 6 ⁇ 10 6 cells/mL, cultured at 37°C at 90 rpm in a 50mm amplitude incubator, 8% CO 2 .
- OPti-PRO SFM medium to prepare transfection reagent and NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid complex (4°C), for example: 1ml cells prepare 40 ⁇ l OPti-PRO SFM add 0.8ug NTD-P2 -6*HIS(GP101150-8) recombinant expression plasmid, mix well for 5 minutes; prepare 40 ⁇ l OPti-PRO SFM, add 3 ⁇ l Expi Fectamine CHO reagent, mix well and leave for 5 minutes, at room temperature for 1-5 minutes, then slowly transfer the solution to a shaker flask and shake , shake the flask gently during the addition.
- the cells were cultured at 37° C., 8% CO 2 in a 50 mm amplitude incubator at 90 rpm. 18-22 hours after transfection, the Enhancer and ExpiCHO Feed were added and the standard protocol was performed.
- B. Electrophoresis: Mix NTD-P2-6*HIS(GP101150-8) purified protein with loading buffer (NTD-P2-6*HIS(GP101150-8) purified protein: 5x loading buffer 4:1 ), boiled for 5 minutes, and sampled. First of all 100V constant voltage electrophoresis, you can see that the Marker gradually becomes a thin line.
- C. Transfer membrane Pry off the glass plate and carefully remove the gel. The PVDF membrane was activated with methanol for 30 s and cut into the same size as the filter paper and soaked in the transfer solution. From bottom to top: filter paper-PVDF membrane-gel-filter paper. Be careful not to have air bubbles between the layers, especially between the glue and the membrane. Pour all the transfer solution in the plate into the transfer box. 20V constant voltage transfer, 50KDa protein needs 20min. The higher the molecular weight, the longer the time required. D. Antibody Incubation.
- ECL color development Each piece of PBST membrane was mixed with 1 mL of solution A and 10 ⁇ L of solution B and dropped on the membrane. After 5 minutes, it was photographed and recorded in the chemiluminescence imaging system, as shown in Figure 2.
- Signal peptide addition MFVFLVLLPLVS (SEQ ID NO: 75), vector selection: pcdna3.1(+). Codon optimization was performed according to the host CHO cells.
- the primers of NTD-RBD-foldon-8*HIS were amplified by PCR to obtain fragment PCR products, which were recombined into the target vector pcdna3.1(+) (BamHI-XhoI restriction vector) by multi-segment recombination to obtain NTD- RBD-foldon-8*HIS recombinant expression plasmid.
- the reaction system is shown in Table 5.
- Transformation method Pipette 1-3 ⁇ l of the NTD-RBD-foldon-8*HIS recombinant expression plasmid with a concentration of about 100 ng/ ⁇ l into about 100 ⁇ l of competent cells, gently shake and rotate to mix, and place on ice for 3 minute. Do not shake in a 42°C water bath for 90s; place in an ice bath for about 3 minutes; add 500-800 ⁇ l of 37°C pre-warmed LB medium to each tube, and shake gently at 37°C at 200 rpm for 40 minutes.
- Verification of NTD-RBD-foldon-8*HIS recombinant expression plasmid (1) Prepare agar plates containing corresponding resistance. (2) Take 100 ⁇ l of bacterial liquid and spread it on an agar plate containing the corresponding resistance. Gently spread the bacteria on the surface of the plate with a sterile glass spreader, and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours to form colonies. (4) Pick the bacteria from the plate, shake the bacteria at 37°C at 250 rpm for 14 hours, use the bacterial solution for PCR identification, and send the positive cloned bacterial solution for sequencing.
- PCR reaction adopts 20 ⁇ L system: primer 0.5 ⁇ L, template bacteria 2 ⁇ L of buffer solution, 0.5 ⁇ L of polymerase buffer, 3 ⁇ L of buffer, and 14 ⁇ L of ddH 2 O. Cycling parameters: pre-denaturation at 96 °C for 3 min; 95 °C for 15 s, 58 °C for 15 s, 72 °C for 20 s, 23 cycles, and a final extension at 72 °C for 1 min.
- the positive clones were screened by bacterial liquid PCR method, and the obtained positive bacterial liquid was shaken at 37°C to extract plasmids and sequenced.
- the correct plasmids were sequenced and compared, and double digested with BamHI--XhoI to obtain two fragments of 1761bp and 5372bp.
- NTD-RBD-foldon-8*HIS recombinant expression plasmid was extracted.
- 1% E. coli cells (stbl3) containing the recombinant expression plasmid of NTD-RBD-foldon-8*HIS were added to 2 ml of LB medium and cultured overnight at 37°C with shaking. After cells were treated, 100 ug of plasmid was extracted using a plasmid extraction kit. The correct plasmids were sequenced and compared, and double-enzyme digestion was performed with BamHI--XhoI to obtain two fragments of 1761bp and 5372bp.
- Day 0 Transfect cells, measure viable cell density and viability, the cell density should reach 7 ⁇ 10 6 –10 ⁇ 10 6 cells/mL, and the viability reaches 95–99% for transfection, fresh cells expressing culture base, preheated to 37°C, cells were diluted to a final density of 6 ⁇ 10 6 cells/mL, cultured at 37°C at 90 rpm in a 50mm amplitude incubator, 8% CO 2 .
- OPti-PRO SFM medium to prepare transfection reagent and NTD-RBD-foldon-8*HIS recombinant expression plasmid complex (4°C), for example: 1ml cells prepare 40ul OPti-PRO SFM add 0.8ug NTD-RBD-foldon- 8*HIS recombinant expression plasmids were mixed and placed for 5 minutes; 40ul of OPti-PRO SFM was prepared and 3ul of Expi Fectamine CHO reagent was added and mixed for 5 minutes, at room temperature for 1-5 minutes, and then the solution was slowly transferred to a shaker and shaken. Shake the bottle gently.
- the cells were placed in a 50 mm amplitude incubator at 90 rpm for incubation at 37°C, 8% CO 2 . 18-22 hours after transfection, add Enhancer and ExpiCHO Feed, and perform standard experimental protocols, for example: 1ml cells add 6ul Enhancer and 0.24ml ExpiCHO Feed, and place cells in a 50mm amplitude incubator at 90rpm for 37°C incubation, 8% CO 2 . Collected 8 days after transfection for subsequent purification.
- Protein purification The culture medium was centrifuged, the supernatant was added with a Ni column, incubated with shaking for 2 hours, and purified by affinity chromatography through a gravity empty column column.
- Equilibration buffer "PBS”, pH7.4, wash 10CV; Wash buffer: “PBS”, pH7.4 with 20mM imidazole, wash 10CV; Elution buffer: “PBS”, pH7.4 with 500mM imidazole, elution 1CV, repeated 5 times to obtain NTD-RBD-foldon-8*HIS purified protein, the results are shown in Table 6.
- B. Electrophoresis: Mix NTD-RBD-foldon-8*HIS purified protein with loading buffer (NR-foldon-8*HIS purified protein: 5x loading buffer 4:1), boil for 5min, and perform spotting . First of all 100V constant voltage electrophoresis, you can see that the Marker gradually becomes a thin line.
- RBD protein 0.04 mg/mL RBD protein, 0.04 mg/mL NTD protein; 2% to 15% (w/v) sucrose; 0.01% to 0.05% (w/v) Tween 80; and 5 mM pH 5.0 to 7.0 to 25mM histidine buffer, dispensed into 2ml vials, 0.5ml per tube, and placed in a lyophilizer for freeze-drying.
- RBD-P2 protein 0.04 mg/mL RBD-P2 protein, 0.04 mg/mL NTD-P2 protein; 2% to 15% (w/v) sorbitol; 0.01% to 0.05% (w/v) polysorbate 20; and 5mM to 20mM histidine buffer at pH 5.5 to 7.0, dispensed into 2ml vials, 0.5ml per tube, and lyophilized in a lyophilizer.
- RBD protein 0.04 mg/mL RBD protein, 0.04 mg/mL NTD-P2 protein; 2% to 15% (w/v) sucrose; 0.01% to 0.05% (w/v) Tween 80; and pH 4.5 to 5.5 of 5mM to 25M succinate buffer, dispensed into 2ml vials, 0.5ml per tube, and placed in a lyophilizer for freeze-drying.
- RBD-P2 protein 0.04 mg/mL RBD-P2 protein, 0.04 mg/mL NTD protein; 2% to 15% (w/v) sorbitol; 0.01% to 0.05% (w/v) polysorbate 20; and pH of 5.5 to 7.0 of 5mM to 20mM histidine buffer, dispensed into 2ml vials, 0.5ml per tube, and placed in a lyophilizer for freeze-drying.
- immunogenic composition of any one of embodiments 8-11 further comprising an aluminum hydroxide adjuvant at a concentration of 1 mg/mL.
- CpG1018 adjuvant was added to the immunogenic composition described in any one of Examples 8-11 at a concentration of 6 mg/mL, and CpG adjuvant (CpG was synthesized by Shanghai Sangon Biotechnology Company according to the sequence of CpG1018).
- immunogenic composition of any one of embodiments 8-11 further comprising an adjuvant, eg, an adjuvant vial of 0.5 ml, 50 ⁇ g of MPL, 500 ⁇ g of aluminum hydroxide, 150 mM NaCl, 8 mM bismuth Disodium hydrogen phosphate water, add water for injection to 0.5ml.
- an adjuvant eg, an adjuvant vial of 0.5 ml, 50 ⁇ g of MPL, 500 ⁇ g of aluminum hydroxide, 150 mM NaCl, 8 mM bismuth Disodium hydrogen phosphate water, add water for injection to 0.5ml.
- the immunogenic composition of any one of embodiments 8-11, further comprising an adjuvant, for example, the adjuvant bottle is 0.25ml, and the components include 10.69mg of squalene and 11.86mg of alpha-tocopherol , 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na 2 HPO 4 , 0.09 mg of KH 2 PO 4 and water for injection.
- the composition further comprises an adjuvant, for example, the adjuvant bottle is 0.5 ml, and the components include 50 ⁇ g of MPL, 50 ⁇ g of QS-21, and 1 mg of DOPC two.
- the immunogenic composition of any one of Embodiments 8-11, further comprising an adjuvant, for example, the adjuvant bottle is 0.5ml, and the components include 9.75mg squalene, 1.175mg Span 85, 1.175mg Tween 80, 0.66 mg trisodium citrate dihydrate and 0.04 mg citric acid monohydrate, and water for injection.
- mice The antigenic protein combined with various adjuvants was used to immunize mice, and the level of neutralizing antibody induced by the protein was determined by ELISA assay. The grouping and immunization schedule of mice are shown in Table 8.
- NTD protein sequence is 1-291 amino acids in SEQ ID NO: 20
- the RBD sequence is 1-223 amino acids in SEQ ID NO: 1, Al(OH) 3 adjuvant, CpG adjuvant, CpG+Al(OH) 3.
- concentration of adjuvant was referred to in Example 12
- the composition of AS01 was referred to in Example 15
- the composition of AS04 was referred to in Example 13.
- the test results after 28 days after immunization of mice are shown in Table 9. The results showed that RBD+NTD induced stronger levels of neutralizing antibodies.
- the antigen protein was used together with MF59 adjuvant to immunize Japanese white rabbits, and the level of neutralizing antibody induced by the protein was determined by ELISA.
- the grouping and immunization schedule of Japanese white rabbits are shown in Table 10.
- NTD-P2 The sequence of NTD-P2 is shown in SEQ ID NO: 20, the sequence of RBD-P2 is shown in SEQ ID NO: 1, the sequence of NTD-foldon is shown in SEQ ID NO: 22, the sequence of RBD-foldon is shown in SEQ ID NO: 3, the sequence of NTD- The sequence of ferritin LC is shown in SEQ ID NO: 23, the sequence of RBD-ferritin is referred to SEQ ID NO: 5, the sequence of NTD-HBsAg is referred to SEQ ID NO: 25, the sequence of RBD-HBsAg is referred to SEQ ID NO: 6, the adjuvant MF59 See Example 16.
- NTD-RBD-P2 refers to the sequence of SEQ ID NO: 40, but there is no HIS tag at the C-terminus
- sequence of NTD-RBD-foldon refers to the sequence of SEQ ID NO: 41, but there is no HIS tag at the C-terminus
- NTD-RBD-P2- The sequence of foldon refers to the sequence of SEQ ID NO: 42, but there is no HIS tag at the C-terminus
- the sequence of NTD-RBD-ferritin refers to the sequence of SEQ ID NO: 43
- the sequence of NTD-RBD-HBsAg refers to the sequence of SEQ ID NO: 44.
- NTD-ferritin+RBD-ferritin MF59 10+10) ⁇ g 128 5 NTD-HBsAg+RBD-HBsAg MF59 (10+10) ⁇ g 128 6 NTD-RBD-P2 MF59 20 ⁇ g 456.1 7 NTD-RBD-foldon MF59 20 ⁇ g 1024 8 NTD-RBD-P2-foldon MF59 20 ⁇ g 1024 9 NTD-RBD-ferritin MF59 20 ⁇ g 512 10 NTD-RBD-HBsAg MF59 20 ⁇ g 456.1
- the antigenic protein was used to immunize rhesus monkeys together with different adjuvants, and the level of neutralizing antibody induced by the protein was determined by ELISA assay.
- the grouping and immunization plan of rhesus monkeys are as follows, and the positive control is the inactivated vaccine of the new crown.
- the detection results of rhesus monkeys 28 days after immunization are shown in Table 12.
- NTD and RBD were selected as candidate antigens.
- the immunogenicity of NTD alone, RBD alone, co-immunization of NTD and RBD, NTD-RBD fusion protein (hereinafter referred to as NR), and NTD-RBD-foldon fusion protein (hereinafter referred to as NR-foldon) was detected, and the results are shown in Table 14. .
- Pseudovirus types prototype strain (WIV04-1), main circulating strain (D614G); VOC mutant strains: British mutant strain (Alpha, B.1.1.7), South African mutant strain (Beta, B.1.351) , Brazil mutant (Gamma, P.1), Indian mutant (Delta, B.1.617.2); VOI mutant strain: Peru mutant (Lambda, C.37), the same below.
- mice were immunized with Al(OH) 3 , BFA01, BFA02, BFA03, BFA04, CpG/Al(OH) 3 and NR-foldon antigen, and neutralizing antibodies and antigen-specific IgG antibodies in serum were detected.
- the results of the study showed that among the candidate adjuvants, the BFA03 adjuvant had the best immune effect and was significantly better than the traditional aluminum adjuvant (aluminum hydroxide adjuvant), as well as the compound adjuvant system based on the aluminum salt adjuvant.
- mice were immunized with 1/10 and 1/5 human doses (ie, 4 ⁇ g and 8 ⁇ g) of NR-foldon antigen in combination with 1/5 or 1/10 human doses of BFA03 adjuvant, respectively.
- the results showed that under the premise of a fixed antigen dose, the neutralizing antibody titer produced by 1/10HD adjuvant immunization was lower than that of 1/5HD adjuvant, and there was no statistical difference between the groups. When appropriate, a stronger immune response can be induced.
- Embodiment 23 Detection of immune effect of different doses in rabbit experiment
- the GMT results of neutralizing antibody titers produced by immunizing different species of animals with ReCOV vaccine are as follows. All strains have good neutralization effect.
- NTD-RBD-foldon (Gamma mutant) as shown in SEQ ID NO:85 adds signal peptides MGWSCIILFLVATATGVHS (SEQ ID NO:91) and 6xHis; NTD-RBD-foldon as shown in SEQ ID NO:79 ( Beta mutant strain) was added with the signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 91) and 6 ⁇ His, the full-length amino acid sequence was codon-optimized by Wuxi WuXi Biologics Co., Ltd., and the whole gene was synthesized by PCR to obtain NTD-RBD- DNA fragments of foldon-6xHis (Gamma mutant) and NTD-RBD-foldon-6xHis (Beta mutant).
- Transform host bacteria (1) Pipette 1-3 ⁇ L of the recombinant expression plasmid of NTD-RBD-foldon-6 ⁇ His (Gamma mutant strain) or NTD-RBD-foldon-6 ⁇ His (Beta mutant strain) at a concentration of 100ng/ ⁇ L Add to 100 ⁇ L of E.coli Top10 competent cells, shake gently to mix, and place on ice for 3 minutes. (2) Do not shake the water bath at 42°C for 90s. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 ⁇ L of pre-warmed LB medium at 37°C to each tube, and shake gently at 37°C at 200 rpm for 40 minutes.
- Extraction and sequencing of recombinant expression plasmids (1) Prepare an agar plate containing 100 ⁇ g/mL ampicillin. (2) After culturing 100 ⁇ L of bacterial liquid, gently spread the LB agar plate (containing 100 ⁇ g/mL ampicillin) with a sterile glass spreader, and incubate the plate at 37° C. for 15 minutes. (3) Invert the plate and culture at 37°C for 12 to 16 hours to form colonies. (4) Pick a single clone from the plate and inoculate it into 300mL LB medium to expand the culture, and use the NucleoBond Xtra Maxi EF kit to prepare the plasmid in large quantities according to the instructions. The target gene was verified by sequencing, and the sequencing results were consistent with the designed genome sequence.
- CHO cells (ExpiCHO from Thermo, Inc.) were subcultured and expanded until the cell density reached 4 ⁇ 10 6 to 6 ⁇ 10 6 cells/mL.
- Day -1 of transfection Adjust cell density to 3 ⁇ 10 6 to 4 ⁇ 10 6 cells/mL and allow cells to grow overnight.
- Day 0 Transfect cells, measure the viable cell density and viability, the cell density should reach 7 ⁇ 10 6 to 10 ⁇ 10 6 cells/mL, and the viability is 95 to 99% for transfection.
- Fresh cell expression medium preheated to 37°C, diluted cells to a final density of 6 ⁇ 106 cells/mL, cultured at 37°C at 90rpm in a 50mm amplitude incubator, 8% CO 2 .
- Use OptiPRO TM SFM medium to prepare transfection reagent and NTD-RBD-foldon-6 ⁇ His (Gamma mutant/Beta mutant) recombinant expression plasmid complex (4°C), eg: 1mL CHO cells prepare 40 ⁇ L OptiPRO TM SFM addition 0.8 ⁇ g NTD-RBD-foldon-6 ⁇ His (Gamma mutant/Beta mutant) recombinant expression plasmid, mix well for 5 minutes; prepare 40 ⁇ L OptiPROTM SFM, add 3 ⁇ L ExpiFectamineTM CHO reagent, mix well for 5 minutes, at room temperature for 1 ⁇ 5 minutes, then slowly transfer the solution to the shake flask and shake gently during the addition.
- the cells were placed in a 50 mm amplitude incubator at 90 rpm for incubation at 37°C, 8% CO 2 . 18-22 hours after transfection, the Enhancer and ExpiCHO Feed were added and the standard protocol was performed.
- IMAC immobilized metal ion affinity chromatography
- SEC size exclusion chromatography
- the cell lysis supernatant was directly loaded on the column, and the solution A (20mmol/L PB, pH7.4, 0.15mol/L NaCl) and solution B (20mmol/L PB, pH7.4, 0.15mol/L NaCl, 0.5mol/L imidazole) was used as the mobile phase for gradient elution (the concentration of solution B was increased from 4% (volume) to 100% (volume)).
- Collect target fractions and proceed to SEC separation.
- HiLoad 26/60 Superdex-200 pre-grade gel column (GE, USA) was used.
- the purified protein of NTD-RBD-foldon-6 ⁇ His (Gamma mutant strain/Beta mutant strain) was obtained.
- the protein purification results are shown in Table 21.
- the purity of the purified target protein can reach 93.6% (Gamma mutant strain) 95% ( Beta mutants).
- B. Electrophoresis: Mix the purified protein of NTD-RBD-foldon (Gamma mutant/Beta mutant) with the loading buffer (NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein: 5 ⁇ loading buffer liquid 4:1), boil for 5 minutes, and perform spotting. First of all 100V constant voltage electrophoresis, you can see that the Marker gradually becomes a thin line.
- Antibody incubation remove the PVDF membrane after transfer, and you can see that the Marker is transferred to the membrane.
- the primary antibody (anti-His Mab) was diluted with 5% nonfat dry milk (1 g nonfat dry milk, 100 mL TBS), incubated in a small box, and reacted in a decolorizing shaker at 4°C for 2 hours.
- the PVDF membrane was taken out and rinsed with PBST for 4 ⁇ 10 min.
- the secondary antibody (goat anti-mouse) was also diluted with 5% nonfat dry milk and incubated for 1 hour.
- NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein 50 ⁇ g of NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein, 1% (w/v) sucrose; 2% (w/v) glycine; 0.02% (w/v) Tween 80; and pH 7 .5 of 10 mM PB buffer (Na 2 HPO 4 , NaH 2 PO 4 ), dispensed into 2 mL vials, 0.5 mL per tube, and placed in a lyophilizer for freeze-drying.
- PB buffer Na 2 HPO 4 , NaH 2 PO 4
- NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein 50 ⁇ g of NTD-foldon (Beta mutant/Gamma mutant) purified protein
- 1% (w/v) sucrose 50 ⁇ g of NTD-foldon (Beta mutant/Gamma mutant) purified protein
- 1% (w/v) sucrose 50 ⁇ g of NTD-foldon (Beta mutant/Gamma mutant) purified protein
- 1% (w/v) sucrose 2% (w/ v) Glycine; 0.02% (w/v) Tween 80
- 10 mM PB buffer Na 2 HPO 4 , NaH 2 PO 4
- NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein 50 ⁇ g of NTD-RBD-foldon (WIV04-1) purified protein; 1% (w/v) sucrose; 2% (w/v) Glycine; 0.02% (w/v) Tween 80; and 10 mM PB buffer (Na 2 HPO 4 , NaH 2 PO 4 ) at pH 7.5, aliquoted into 2 mL vials, 0.5 mL per tube, lyophilized freeze-drying machine.
- PB buffer Na 2 HPO 4 , NaH 2 PO 4
- NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein 50 ⁇ g of NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein; 135 ⁇ g of HA protein (45 ⁇ g of H1N1 HA protein, 45 ⁇ g of H3N2 HA protein, 45 ⁇ g of B/Washington/02/2019 HA protein ); 1% (w/v) sucrose; 2% (w/v) glycine; 0.02% (w/v) Tween 80; and 10 mM PB buffer (Na 2 HPO 4 , NaH 2 PO ) at pH 7.5 4 ), dispensed into 2mL vials, 0.5mL per tube, and placed in a lyophilizer for freeze-drying.
- the selected HA proteins are shown in Table 23.
- NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein 50 ⁇ g of NTD-RBD-foldon (Gamma mutant/Beta mutant) purified protein; 180 ⁇ g of HA protein (45 ⁇ g of H1N1 HA protein, 45 ⁇ g of H3N2 HA protein, 45 ⁇ g of B/Washington/02/2019 HA protein , 45 ⁇ g of HA protein of B/Phuket/3073/2013); 1% (w/v) sucrose; 2% (w/v) glycine; 0.02% (w/v) Tween 80; and 10 mM pH 7.5 PB buffer (Na 2 HPO 4 , NaH 2 PO 4 ) was dispensed into 2 mL vials, 0.5 mL per tube, and lyophilized in a lyophilizer.
- the selected HA proteins are shown in Table 23.
- immunogenic composition of any one of the preceding embodiments, further comprising an aluminum hydroxide adjuvant at a concentration of 1 mg/mL.
- the immunogenic composition described in any one of the preceding embodiments further comprises adjuvant MF59, the adjuvant bottle is 0.5mL, and the components include 4.5% squalene, 0.5% Tween 80, 0.5% Span85 and water for injection.
- the immunogenic composition described in Example 28 was selected in combination with AS03 adjuvant, and the mice were immunized according to 2/25 human doses, and the immunogenic composition prepared by NTD-RBD-foldon (WIV04-1) protein was combined AS03 adjuvant was used as a control, and the level of induced neutralizing antibody was determined by the new coronavirus pseudovirus neutralizing antibody detection method based on the VSV (vesicular stomatitis virus) system.
- the grouping and immunization schedule of mice are shown in Table 24.
- Antigen NTD-RBD-foldon adjuvant Antigen dose Immunization program immune pathway animal (only) Beta mutant AS03 4 ⁇ g 2 doses, 14d interval intramuscular injection 10 WIV04-1 AS03 4 ⁇ g 2 doses, 14d interval intramuscular injection 10 WIV04-1+Beta mutant strain AS03 (2+2) ⁇ g 2 doses, 14d interval intramuscular injection 10
- the preparation of AS03 adjuvant refers to Example 29.
- serum was obtained by blood sampling from the orbital vein, and the neutralizing antibody titer was detected by the VSV-based pseudovirus detection system.
- the main epidemic strains of the new coronavirus (D614G ) pseudovirus, new coronavirus Alpha mutant strain (B.1.1.7) pseudovirus, new coronavirus Beta mutant strain (501Y.V2) pseudovirus, and new coronavirus Gamma mutant strain (501Y.V3) pseudovirus were neutralized.
- the neutralizing effect of the vaccine on different new coronavirus variant strains, the test results are shown in Table 25.
- the monovalent vaccine using NTD-RBD-foldon (Beta mutant strain) as the immunogen has good immune effect on the four new coronavirus mutant strains, and it can especially be used as a new response to SARS-CoV-2 mutant strain (In particular, the COVID-19 vaccine candidates from South Africa and Gamma mutant strains are more efficient in inducing the production of protective neutralizing antibodies against immune evasion of the new coronavirus mutant strains.
- the bivalent vaccine with NTD-RBD-foldon (Beta mutant) combined with NTD-RBD-foldon (WIV04-1) as the immunogen was compared with the NTD-RBD-foldon (Beta mutant) monovalent vaccine and NTD-RBD-
- the foldon (WIV04-1) monovalent vaccine induces an overall increase in the level of neutralizing antibodies, which not only enhances the effect of the NTD-RBD-foldon (WIV04-1) monovalent vaccine on the Beta mutant pseudovirus, but also enhances the NTD-RBD -Effect of foldon (Beta mutant) monovalent vaccine on the main circulating pseudovirus and British circulating pseudovirus.
- This bivalent vaccine has the characteristics of wider range of action and stronger effect, and is more suitable to play a role as a candidate vaccine against COVID-19.
- the immunogenic composition described in Example 28 was selected in combination with AS03 adjuvant, and the mice were immunized according to the 2/25 human dose, and the level of neutralizing antibodies induced was passed through the new coronavirus pseudovirus based on the VSV (vesicular stomatitis virus) system. Neutralizing antibody detection method.
- the grouping and immunization schedule of mice are shown in Table 26.
- the preparation of AS03 adjuvant refers to Example 29.
- serum was obtained by blood sampling from the orbital vein, and the neutralizing antibody titer was detected by the VSV-based pseudovirus detection system.
- the main epidemic strains of the new coronavirus (D614G ) pseudovirus, new coronavirus Alpha mutant strain (B.1.1.7) pseudovirus, new coronavirus Beta mutant strain (501Y.V2) pseudovirus, and new coronavirus Gamma mutant strain (501Y.V3) pseudovirus were neutralized.
- the neutralization effect of the vaccine on different new coronavirus variant strains, the test results are shown in Table 27.
- mice with NTD-RBD-foldon (Gamma mutant) as antigen-binding AS03 adjuvant induced the production of neutralizing Beta mutant (501Y.V2) pseudovirus with the highest antibody level, and induced the neutralizing Gamma mutant (501Y.V2).
- 501Y.V3 pseudovirus had the second highest level of antibodies, while lower levels of antibodies were induced to neutralize the main circulating strain (D614G) pseudovirus and the Alpha mutant (B.1.1.7) pseudovirus.
- the monovalent vaccine using NTD-RBD-foldon (Gamma mutant strain) as the immunogen is effective against the four new coronavirus mutant strains, and it can be used as a new response to SARS-CoV-2 mutant strains (especially South Africa and Gamma strains). mutant) of the COVID-19 vaccine candidate, which more efficiently induces the production of protective neutralizing antibodies against immune escape from the new coronavirus mutant.
- the bivalent vaccine of NTD-RBD-foldon (Gamma mutant) combined with NTD-RBD-foldon (Beta mutant), or NTD-RBD-foldon (Gamma mutant) combined with NTD is prepared according to the above method - Bivalent vaccine of RBD-foldon (WIV04-1), or NTD-RBD-foldon (Gamma mutant) combined with NTD-RBD-foldon (Beta mutant) and NTD-RBD-foldon (WIV04-1) trivalent Vaccine, or multivalent combination vaccine such as NTD-RBD-foldon (Gamma mutant) combined with NTD-RBD-foldon (Beta mutant), NTD-RBD-foldon (WIV04-1) and influenza hemagglutinin HA May produce higher neutralizing antibody titers.
Abstract
本申请涉及一种或多种融合蛋白,其包含SARS-CoV-2 S(Spike)蛋白的结构域。本申请还涉及了包含融合蛋白的免疫原性组合物及其应用。
Description
本申请涉及生物医药领域,具体的涉及包含SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段和/或SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段的融合蛋白、组合物及其应用。
目前,控制新冠疫情的方式是实施检疫、隔离和保持物理距离。因此迫切需要针对COVID-19的有效疫苗,以减轻与SARS-CoV-2感染相关的死亡率和发病率的巨大负担。
SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)被认为是诱导机体产生中和抗体的最主要的抗原靶区域。RBD作为疫苗能够将机体刺激产生的中和抗体更加聚焦在针对病毒的受体结合,可以提高疫苗的免疫原性和免疫效率。
SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)为病毒S蛋白N端的一段序列,其可与宿主细胞的蛋白或糖蛋白结合,介导病毒入侵宿主细胞,故该段区域可能包含诱导中和抗体产生的表位。
发明内容
本申请提供了一种包含SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段和/或SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段的融合蛋白、组合物及其应用,其具有下列性质的一种或多种:1)融合蛋白具有免疫原性;2)包含融合蛋白的COVID-19亚单位疫苗免疫原性更强,能诱导产生更多的中和抗体。
一方面,本申请提供了一种融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及2)一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
在某些实施方式中,所述RBD源自SARS-CoV-2野生型或其突变体。
在某些实施方式中,所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
在某些实施方式中,所述RBD与野生型SAR-CoV-2的RBD相比,在选自下组的一个或 多个氨基酸位点有突变:K417、L452、T478、E484和N501。
在某些实施方式中,所述RBD包含氨基酸突变K417T/N、L452R、T478K、E484K和N501Y的一种或多种。
在某些实施方式中,所述RBD包含K417T、E484K和N501Y氨基酸突变。
在某些实施方式中,所述RBD包含K417N、E484K和N501Y氨基酸突变。
在某些实施方式中,所述RBD包含SEQ ID NO:18、19、76、83、97-108中任一项所示的氨基酸序列。
在某些实施方式中,所述P2或其功能活性片段包含破伤风毒素的表位肽。
在某些实施方式中,所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
在某些实施方式中,所述P2或其功能活性片段与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
在某些实施方式中,所述RBD或其功能活性片段与所述P2或其功能活性片段在框内融合。
在某些实施方式中,所述foldon结构域或其功能活性片段包含噬菌体T4纤维蛋白C末端的氨基酸残基。
在某些实施方式中,所述foldon结构域或其功能活性片段包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
在某些实施方式中,所述foldon结构域或其功能活性片段与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
在某些实施方式中,所述RBD或其功能活性片段与所述foldon结构域或其功能活性片段在框内融合。
在某些实施方式中,所述铁蛋白或其功能活性片段包含粉纹夜蛾铁蛋白或其功能活性片段。
在某些实施方式中,所述粉纹夜蛾铁蛋白或其功能活性片段包括粉纹夜蛾铁蛋白的重链或轻链。
在某些实施方式中,所述粉纹夜蛾铁蛋白的重链包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中,所述粉纹夜蛾铁蛋白的轻链包含SEQ ID NO:71所示的氨基酸序列。
在某些实施方式中,所述铁蛋白或其功能活性片段包含幽门螺杆菌铁蛋白或其功能活性片段。
在某些实施方式中,所述铁蛋白或其功能活性片段包含SEQ ID NO:70-72中任一项所示的氨基酸序列。
在某些实施方式中,所述铁蛋白或其功能活性片段与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
在某些实施方式中,所述RBD或其功能活性片段与所述铁蛋白或其功能活性片段在框内融合。
在某些实施方式中,所述乙肝表面抗原或其功能活性片段包含SEQ ID NO:73所示的氨基酸序列。
在某些实施方式中,所述乙肝表面抗原或其功能活性片段与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
在某些实施方式中,所述RBD或其功能活性片段与所述乙肝表面抗原或其功能活性片段在框内融合。
在某些实施方式中,所述的融合蛋白包含SEQ ID NO:1-17、80和86中任一项所示的氨基酸序列。
另一方面,本申请提供了一种融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段;以及2)一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
在某些实施方式中,所述NTD源自SARS-CoV-2野生型或其突变体。
在某些实施方式中,所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
在某些实施方式中,所述NTD包含在选自下组的一个或多个氨基酸位点的突变:L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246。
在某些实施方式中,所述NTD包含选自下组的一个或多个氨基酸突变:L18F、T19R、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I。
在某些实施方式中,所述NTD包含L18F、D80A、D215G、L242-244del和R246I突变。
在某些实施方式中,所述NTD包含L18F、T20N、P26S、D138Y和R246I氨基酸突变。
在某些实施方式中,所述NTD包含SEQ ID NO:37、38、77、84和109-111中任一项所示的氨基酸序列。
在某些实施方式中,所述P2或其功能活性片段包含破伤风毒素的表位肽。
在某些实施方式中,所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
在某些实施方式中,所述P2或其功能活性片段与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
在某些实施方式中,所述NTD或其功能活性片段与所述P2或其功能活性片段在框内融合。
在某些实施方式中,所述foldon结构域或其功能活性片段包含噬菌体T4纤维蛋白C末端的氨基酸残基。
在某些实施方式中,所述foldon结构域或其功能活性片段包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
在某些实施方式中,所述foldon结构域或其功能活性片段与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
在某些实施方式中,所述NTD或其功能活性片段与所述foldon结构域或其功能活性片段在框内融合。
在某些实施方式中,所述铁蛋白或其功能活性片段包含粉纹夜蛾铁蛋白或其功能活性片段。
在某些实施方式中,所述粉纹夜蛾铁蛋白或其功能活性片段包括粉纹夜蛾铁蛋白的重链或轻链。
在某些实施方式中,所述粉纹夜蛾铁蛋白的重链包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中,所述粉纹夜蛾铁蛋白的轻链包含SEQ ID NO:71所示的氨基酸序列。
在某些实施方式中,所述铁蛋白或其功能活性片段包含幽门螺杆菌铁蛋白或其功能活性片段。
在某些实施方式中,所述铁蛋白或其功能活性片段包含SEQ ID NO:70-72任一项所示的氨基酸序列。
在某些实施方式中,所述铁蛋白或其功能活性片段与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
在某些实施方式中,所述NTD或其功能活性片段与所述铁蛋白或其功能活性片段在框内融合。
在某些实施方式中,所述乙肝表面抗原或其功能活性片段包含SEQ ID NO:73所示的氨基酸序列。
在某些实施方式中,所述乙肝表面抗原或其功能活性片段与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
在某些实施方式中,所述NTD或其功能活性片段与所述乙肝表面抗原或其功能活性片段在框内融合。
在某些实施方式中,所述的融合蛋白包含SEQ ID NO:20-36、81和87中任一项所示的氨基酸序列。
另一方面,本申请提供了一种融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及2)SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
在某些实施方式中,所述的融合蛋白还包括一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
在某些实施方式中,所述RBD源自SARS-CoV-2野生型或其突变体。
在某些实施方式中,所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
在某些实施方式中,所述RBD与野生型SAR-CoV-2的RBD相比,在选自下组的一个或多个氨基酸位点有突变:K417、L452、T478、E484和N501。
在某些实施方式中,所述RBD包含氨基酸突变K417T/N、L452R、T478K、E484K和N501Y的一种或多种。
在某些实施方式中,所述RBD包含K417T、E484K和N501Y氨基酸突变。
在某些实施方式中,所述RBD包含K417N、E484K和N501Y氨基酸突变。
在某些实施方式中,所述RBD包含SEQ ID NO:18、19、76、83、97-108中任一项所示的氨基酸序列。
在某些实施方式中,所述NTD源自SARS-CoV-2野生型或其突变体。
在某些实施方式中,所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
在某些实施方式中,所述NTD包含在选自下组的一个或多个氨基酸位点的突变:L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246。
在某些实施方式中,所述NTD包含选自下组的一个或多个氨基酸突变:L18F、T19R、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I。
在某些实施方式中,所述NTD包含L18F、D80A、D215G、L242-244del和R246I突变。
在某些实施方式中,所述NTD包含L18F、T20N、P26S、D138Y和R246I氨基酸突变。
在某些实施方式中,所述NTD包含SEQ ID NO:37、38、77、84和109-111中任一项所示的氨基酸序列。
在某些实施方式中,所述RBD或其功能活性片段与所述NTD或其功能活性片段直接或间接地相连。
在某些实施方式中,所述RBD或其功能活性片段与所述NTD或其功能活性片段在框内融合。
在某些实施方式中,所述P2或其功能活性片段包含破伤风毒素的表位肽。
在某些实施方式中,所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
在某些实施方式中,所述P2或其功能活性片段与所述RBD或其功能性片段和/或所述NTD或其功能活性片段直接或间接地相连。
在某些实施方式中,所述P2或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
在某些实施方式中,所述P2或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性片段的N端与所述NTD或其功能活性片段的C端直接或间接地相连。
在某些实施方式中,所述foldon结构域或其功能活性片段包含噬菌体T4纤维蛋白C末端的氨基酸残基。
在某些实施方式中,所述噬菌体T4纤维蛋白C末端的氨基酸残基包含SEQ ID NO:78所示的氨基酸序列。
在某些实施方式中,所述foldon结构域或其功能活性片段包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
在某些实施方式中,所述foldon结构域或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
在某些实施方式中,所述foldon结构域或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
在某些实施方式中,所述foldon结构域或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活 性片段的C端直接或间接地相连。
在某些实施方式中,所述foldon结构域或其功能活性片段的N端与所述P2或其功能活性片段的C端直接或间接地相连,所述P2或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活性的C端直接或间接地相连。
在某些实施方式中,所述铁蛋白或其功能活性片段包含粉纹夜蛾铁蛋白或其功能活性片段。
在某些实施方式中,所述粉纹夜蛾铁蛋白或其功能活性片段包括粉纹夜蛾铁蛋白的重链或轻链。
在某些实施方式中,所述粉纹夜蛾铁蛋白的重链包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中,所述粉纹夜蛾铁蛋白的轻链包含SEQ ID NO:71所示的氨基酸序列。
在某些实施方式中,所述铁蛋白或其功能活性片段包含幽门螺杆菌铁蛋白或其功能活性片段。
在某些实施方式中,所述铁蛋白或其功能活性片段包含SEQ ID NO:70-72任一项所示的氨基酸序列。
在某些实施方式中,所述铁蛋白或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
在某些实施方式中,所述铁蛋白或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
在某些实施方式中,所述铁蛋白或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活性片段的C端直接或间接地相连。
在某些实施方式中,所述乙肝表面抗原或其功能活性片段包含SEQ ID NO:73所示的氨基酸序列。
在某些实施方式中,所述乙肝表面抗原或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
在某些实施方式中,所述乙肝表面抗原或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
在某些实施方式中,所述乙肝表面抗原或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活 性片段的C端直接或间接地相连。
在某些实施方式中,所述的融合蛋白包含SEQ ID NO:39-44、79、85和96中任一项所示的氨基酸序列。
另一方面,本申请提供了一种免疫原性组合物,其包含所述的融合蛋白。
在某些实施方式中,所述的免疫原性组合物包含第一组分,所述第一组分包含SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及第二组分,所述第二组分包含融合蛋白。
在某些实施方式中,所述的免疫原性组合物包含第一组分,所述第一组分包含融合蛋白;以及第二组分,所述第二组分包含SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
在某些实施方式中,所述的免疫原性组合物包含第一组分,所述第一组分包含所述的融合蛋白;以及第二组分,所述第二组分包含所述的融合蛋白。
在某些实施方式中,所述的免疫原性组合物,其按下述重量比配制而成:1)第一组分(1-15重量份);和/或2)第二组分(1-15重量份)。
在某些实施方式中,所述的免疫原性组合物,其包含:1)5-60μg第一组分;和/或2)5-60μg第二组分。
另一方面,本申请提供了一种免疫原性组合物,其包含第一组分,所述第一组分包含SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及第二组分,所述第二组分包含SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
在某些实施方式中,所述RBD源自SARS-CoV-2野生型或其突变体。
在某些实施方式中,所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
在某些实施方式中,所述RBD与野生型SAR-CoV-2的RBD相比,在选自下组的一个或多个氨基酸位点有突变:K417、L452、T478、E484和N501。
在某些实施方式中,所述RBD包含氨基酸突变K417T/N、L452R、T478K、E484K和N501Y的一种或多种。
在某些实施方式中,所述RBD包含K417T、E484K和N501Y氨基酸突变。
在某些实施方式中,所述RBD包含K417N、E484K和N501Y氨基酸突变。
在某些实施方式中,所述RBD包含SEQ ID NO:18、19、76、83、97-108中任一项所示 的氨基酸序列。
在某些实施方式中,所述RBD源自SARS-CoV-2野生型或其突变体。
在某些实施方式中,所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
在某些实施方式中,所述NTD包含在选自下组的一个或多个氨基酸位点的突变:L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246。
在某些实施方式中,所述NTD包含选自下组的一个或多个氨基酸突变:L18F、T19R、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I。
在某些实施方式中,所述NTD包含L18F、D80A、D215G、L242-244del和R246I突变。
在某些实施方式中,所述NTD包含L18F、T20N、P26S、D138Y和R246I氨基酸突变。
在某些实施方式中,所述NTD包含SEQ ID NO:37、38、77、84和109-111中任一项所示的氨基酸序列。
在某些实施方式中,所述的免疫原性组合物,其按下述重量比配制而成:1)第一组分(1-15重量份);和/或2)第二组分(1-15重量份)。
在某些实施方式中,所述的免疫原性组合物,其包含:1)5-60μg第一组分;和/或2)5-60μg第二组分。
在某些实施方式中,所述RBD包含SEQ ID NO:18-19、76、83、97-108中任一项所示的氨基酸序列。
在某些实施方式中,所述NTD包含SEQ ID NO:37-38、77、84和109-111中任一项所示的氨基酸序列。
另一方面,本申请提供了一种药物组合物,其包含所述的融合蛋白,或所述的免疫原性组合物,及任选地药学上可接受的赋形剂。
另一方面,本申请提供了一种所述的融合蛋白或所述的免疫原性组合物在制备疫苗中的用途。
在某些实施方式中,所述疫苗用于预防和/或治疗COVID-19。
另一方面,本申请提供了一种所述的融合蛋白或所述的免疫原性组合物,用于治疗和/或预防COVID-19。
另一方面,本申请提供了一种制备COVID-19亚单位疫苗的方法,其包括:
1)提供所述的融合蛋白或所述的免疫原性组合物;以及
2)使1)中所述融合蛋白或免疫原性组合物与药学上可接受的佐剂混合。
另一方面,本申请提供了一种检测SARS-CoV-2中和抗体的方法,其包括:
1)向受试者施用所述的COVID-19亚单位疫苗;以及
2)检测1)中所述受试者在接受所述COVID-19亚单位疫苗后体内产生的中和抗体。
另一方面,本申请提供了一种治疗和/或预防COVID-19的方法,其包括向受试者施用所述的融合蛋白或所述的免疫原性组合物或所述的COVID-19亚单位疫苗。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的,本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地,本申请的附图和说明书中的描述仅仅是示例性的,而非为限制性的。
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下:
图1显示的是用SDS-PAGE电泳与WB验证RBD-P2-6*HIS蛋白的结果图。
图2显示的是用SDS-PAGE电泳与WB验证NTD-P2-6*HIS(GP101150-8)蛋白的结果图。
图3显示的是用SDS-PAGE电泳与WB验证NTD-RBD-foldon-8*HIS蛋白的结果图。
图4显示的是NTD-RBD-foldon-6×His(Gamma突变株)测序结果氨基酸序列比对。
图5显示的是NTD-RBD-foldon(Gamma突变株)蛋白的尺寸排阻层析图谱。
图6显示的是SDS-PAGE电泳与Western blot验证NTD-RBD-foldon(Gamma突变株)蛋白结果图。
图7显示的是NTD-RBD-foldon-6×His(Beta突变株)测序结果氨基酸序列比对。
图8显示的是NTD-RBD-foldon(Beta突变株)蛋白的尺寸排阻层析图谱。
图9显示的是SDS-PAGE电泳与Western blot验证NTD-RBD-foldon(Beta突变株)蛋白结果图。
以下由特定的具体实施例说明本申请发明的实施方式,熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中,术语“融合蛋白”通常是指通过基因工程技术得到的具有生物学功能活性的蛋白分子。在本申请中,所述融合蛋白可以是RBD或其功能活性片段与下列任一序列组成的融合蛋白:P2或其功能活性片段、foldon结构域或其功能活性片段、铁蛋白或其功能活性片段、乙肝表面抗原HBsAg或其功能活性片段。在本申请中,融合蛋白可以是NTD或其功能活性片段与下列任一序列组成的融合蛋白:P2或其功能活性片段、foldon结构域或其功能活性片段、铁蛋白或其功能活性片段、乙肝表面抗原HBsAg或其功能活性片段。在本申请中,融合蛋白可以是NTD或其功能活性片段和RBD或其功能活性片段组成的融合蛋白。在本申请中,融合蛋白可以是NTD或其功能活性片段、RBD或其功能活性片段与下列任一片段组成的融合蛋白:P2或其功能活性片段、foldon结构域或其功能活性片段、铁蛋白或其功能活性片段、乙肝表面抗原HBsAg或其功能活性片段。
GISAID将毒株hCoV-19/WIV04/2019(WIV04)的全基因组序列(EPI_ISL_402124)做为正式的参考序列。一般将WIV04毒株定义为野生株或原始株。在本申请中,术语“SARS-CoV-2野生型”通常指WIV04毒株。
在本申请中,术语“P2”通常是指破伤风毒素的表位肽。例如,P2可以是破伤风毒素的表位肽830到844的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽830到845的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽829到844的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽829到844的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。例如,P2可以是破伤风毒素的表位肽830到845的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。例如,P2可以是破伤风毒素的表位肽830到844的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。
在本申请中,术语“foldon结构域”通常是指在噬菌体T4纤维蛋白的C末端的残基。在本申请中,foldon结构域可以是噬菌体T4纤维蛋白的C末端的27个残基或突变体。在本申请中,foldon结构域可以是噬菌体T4纤维蛋白的C末端的27个残基截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的截短体或增长体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。
在本申请中,术语“铁蛋白”、“ferritin”通常是指粉纹夜蛾铁蛋白或幽门螺旋杆菌铁蛋 白。在本申请中,粉纹夜蛾铁蛋白具有重链和轻链(ferritin LC和ferritin HC)。在本申请中,幽门螺旋杆菌铁蛋白具有单链结构。在本申请中,铁蛋白可以是粉纹夜蛾或幽门螺旋杆菌铁蛋白的突变体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。在本申请中,“粉纹夜蛾铁蛋白的轻链”、“LC”可以与“ferritin LC”互换使用。在本申请中,“粉纹夜蛾铁蛋白的重链”、“HC”可以与“ferritin HC”互换使用。
在本申请中,术语“乙肝表面抗原(HBsAg)”通常是指乙肝病毒最外层包膜中的一种外壳蛋白。例如,乙肝表面抗原的氨基酸序列可以为NCBI中的蛋白序列检索号AAA45524、ANJ76941、CAA24234或AAC34729对应的序列,也可以适宜地截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸,或有蛋白的突变,例如缺失、置换或插入一个或多个氨基酸。
在本申请中,术语“功能活性片段”通常是指与RBD、NTD、P2、foldon结构域、铁蛋白、乙肝表面抗原HBsAg具有相似生物学活性的片段。
在本申请中,术语“免疫原性组合物”通常是指一种亚单位组合物。在本申请中,亚单位组合物是在将组分混合形成抗原性组合物之前,其中组分已经被分离并纯化到至少50%、至少60%、70%、80%、90%纯度的组合物。例如,亚单位组合物可以是水溶性蛋白的水溶液。例如,所述亚单位组合物可包含洗涤剂。例如,亚单位组合物可包含非-离子、两性离子或离子洗涤剂。例如,亚单位组合物可包含脂类。在一些情形中,免疫原性组合物可包括RBD或其功能活性片段和NTD或其功能活性片段。在一些情形中,免疫原性组合物可包括RBD或其功能活性片段和包含NTD或其功能活性片段的融合蛋白。在一些情形中,免疫原性组合物可包括包含RBD或其功能活性片段的融合蛋白和NTD或其功能活性片段。在一些情形中,免疫原性组合物可包括包含RBD或其功能活性片段的融合蛋白和包含NTD或其功能活性片段的融合蛋白。在本申请中,所述免疫原性组合物还可包括佐剂。例如,所述的佐剂可以包括铝盐(例如氢氧化铝凝胶(alum)或磷酸铝),但也可以是钙盐、铁盐或锌盐,或者可以是酰化酪氨酸或酰化糖、阳离子或阴离子衍生化多糖或聚磷腈的不溶性悬浮液。例如,所述的免疫原性组合物还可以选择为Th1型应答优先诱导物。例如,Th1型应答优先诱导物可以包括单磷酰脂质A或其衍生物。例如,所述佐剂可以是单磷酰脂质A(例如,3-脱-O-酰化单磷酰脂质A(3D-MPL))和铝盐的组合。一种佐剂增强系统可以包括单磷酰脂质A和皂苷衍生物的组合,特别是WO94/00153公开的QS21和3D-MPL的组合,或者如WO96/33739公开的用胆固醇将QS21猝灭从而使反应原性较弱的一种组合物。例如,所述佐剂还可以是 WO95/17210中所述的佐剂,其在水包油乳剂中含有QS21、3D-MPL和生育酚。例如,所述佐剂可以是吐温80、山梨糖醇三油酸酯和角鲨烯混合后于高压条件下进行微流化形成的均一小滴状乳液。例如,所述佐剂可包含寡核苷酸的未甲基化CpG(WO96/02555)。
在本申请中,术语“重量份”通常是指免疫原性组合物的第一组分和第二组分的重量比。在本申请中,免疫原性组合物每剂量可含有如下成份:第一组分5-60μg和第二组分5-60μg。例如,免疫原性组合物可以包含5μg、10μg、20μg、30μg或40μg的第一组分。例如,免疫原性组合物可以包含5μg、10μg、20μg、30μg或40μg的第二组分。
在本申请中,术语“RBD”是指SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域。在本申请中,所述RBD可为SARS-CoV-2刺突蛋白(S蛋白)310-560氨基酸之间的肽段或其突变体,也可以截短N端或C端1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、20、25或30个氨基酸。在本申请中,所述RBD可为SARS-CoV-2刺突蛋白(S蛋白)319-541氨基酸之间的肽段或其突变体,也可以适宜地截短N端或C端1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、20、25或30个氨基酸。在本申请中,所述RBD可为SARS-CoV-2刺突蛋白(S蛋白)331-524氨基酸之间的肽段或其突变体,也可以适宜地截短N端或C端1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、20、25或30个氨基酸。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。
在某些实施方式中,所述RBD可以包含SARS-CoV-2(Gamma突变体)的RBD。
在某些实施方式中,所述RBD可以包含SARS-CoV-2(Beta突变体)的RBD。
在某些实施方式中,所述RBD可以包含SARS-CoV-2(WIV04-1)的RBD。
在本申请中,术语“NTD”即SARS-CoV-2刺突蛋白(S蛋白)N端的结构域。在本申请中,所述NTD可以为SARS-CoV-2刺突蛋白(S蛋白)13-353氨基酸之间的肽段或其突变体,也可以适宜地截短N端或C端1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、20、25或30个氨基酸。例如,所述NTD可以为SARS-CoV-2刺突蛋白(S蛋白)13-303氨基酸之间的肽段或其突变体。在本申请中,所述NTD可以为SARS-CoV-2刺突蛋白(S蛋白)14-304氨基酸之间的肽段或其突变体。在本申请中,所述NTD可以为SARS-CoV-2刺突蛋白(S蛋白)18-353氨基酸之间的肽段或其突变体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。在某些实施方式中,所述NTD可以包含SARS-CoV-2(Gamma突变体)的NTD。在某些实施方式中,所述NTD可以包含SARS-CoV-2(Beta突变体)的 NTD。在某些实施方式中,所述NTD可以包含SARS-CoV-2(WIV04-1)的NTD。
在本申请中,术语“S蛋白”,也称为“Spike蛋白”或“刺突蛋白”,通常是指冠状病毒的衣壳表面糖蛋白。SARS-COV-2通过S蛋白与ACE2受体结合并侵入细胞。S蛋白由1213个氨基酸组成,含有一个跨膜区,包含来自冠状病毒的衣壳表面糖蛋白从N端起或从第14位氨基酸起至少到1213氨基酸片段,或者来自其它SARS病毒的相应区域。S1蛋白是S蛋白的亚单位1,主要指从N端或第14位氨基酸起到第685氨基酸片段。
在本申请中,所述突变体可以包含目前已知的任意一种或几种SAR-CoV-2突变株(突变体)。例如,所述Gamma突变株可以参考GenBank登录号为QRN46961.1的B.1.1.28谱系的SARS-CoV-2表面糖蛋白氨基酸序列。例如,所述Gamma突变株可以参考GenBank登录号为QLF80256.1的B.1.1.28谱系的SARS-CoV-2表面糖蛋白氨基酸序列。例如,所述Gamma突变株可以参考GenBank登录号为QVE55289.1的P.1谱系的SARS-CoV-2表面糖蛋白氨基酸序列。例如,所述Gamma突变株可以为Gamma突变株501Y.V3,其表面糖蛋白氨基酸序列可以如SEQ ID NO:88所示。
例如,所述Beta突变株可以参考GenBank登录号为QUA12570.1的B.1.351谱系的SARS-CoV-2表面糖蛋白氨基酸序列。例如,所述Beta突变株可以参考GenBank登录号为QIZ15537.1的B.1谱系的SARS-CoV-2表面糖蛋白氨基酸序列。例如,所述Beta突变株可以参考GenBank登录号为QVI03430.1的B.1.351谱系的SARS-CoV-2表面糖蛋白氨基酸序列。例如,所述Beta突变株可以为501Y.V2Beta突变株,其表面糖蛋白氨基酸序列如SEQ ID NO:82所示。
在本文中所使用的术语“血凝素/血凝素蛋白/HA”通常是指可使红血球凝集的抗体或其他物质,在流感病毒、麻疹病毒(以及许多其他细菌和病毒)表面等均能找到,可附着于不同动物的红血球,而使红血球凝集,严重时可致死。在本文中是指流感病毒的表面糖蛋白hemagglutinin(HA)。每年WHO官网均会公布当年流感序列的预测,本文参考的是2021-2022年度的北半球的流感序列,即GISAID官网可查询的流感病毒A型(H1N1)EPI1661231|HA|A/Wisconsin/588/2019|EPI_ISL_404460,A型(H3N2)EPI1843589|HA|A/Cambodia/e0826360/2020|EPI_ISL_944639,B型EPI1394970|HA|B/Washington/02/2019|EPI_ISL_347829和B型EPI529345|HA|B/PHUKET/3073/2013|EPI_ISL_161843。
在本申请中,术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下,也表示“为”、“由……组成”的含义。
在本申请中,术语“左右”通常是指在指定数值以上或以下0.5%-10%的范围内变动, 例如在指定数值以上或以下0.5%、1%、1.5%、2%、2.5%、3%、3.5%、4%、4.5%、5%、5.5%、6%、6.5%、7%、7.5%、8%、8.5%、9%、9.5%、或10%的范围内变动。
在本申请中,术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子,其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体,以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常,通过培养包含所述载体的合适的宿主细胞,所述载体可以产生期望的表达产物。
在本申请中,术语“宿主细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体,或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞,细胞系或细胞培养物。所述宿主细胞可以包括单个宿主细胞的子代。由于天然的,意外的或故意的突变,子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同,但能够表达本申请所述的融合蛋白即可。所述宿主细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述宿主细胞可以是原核细胞(例如大肠杆菌),也可以是真核细胞(例如酵母细胞,例如COS细胞,中国仓鼠卵巢(CHO)细胞,HeLa细胞,HEK293细胞,COS-1细胞,NS0细胞或骨髓瘤细胞)。在一些实施方案中,所述宿主细胞是哺乳动物细胞。例如,所述哺乳动物细胞可以是CHO细胞。
发明详述
融合蛋白
一方面,本申请提供了一种融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及2)一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
在本申请中,所述RBD或其功能活性片段可以来源于SARS-CoV-2野生型S蛋白的RBD,也可以来源于SARS-CoV-2突变体S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以在K417、L452、T478、E484和N501中的一个或多个氨基酸位点处包含氨基酸突变。
在本申请中,所述RBD或其功能活性片段可以包含Gamma突变株S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以包含K417T、L452R、T478K、E484K和N501Y中的一个或多个氨基酸突变。例如,所述RBD可以包含SEQ ID NO:83所示的氨基 酸序列。在本申请中,所述RBD或其功能活性片段可以包含Beta突变株S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以包含K417N、E484K和N501Y中的一个或多个氨基酸突变。例如,所述RBD可以包含SEQ ID NO:76所示的氨基酸序列。
在本申请中,所述P2或其功能活性片段可以包含破伤风毒素的表位肽。
例如,P2可以是破伤风毒素的表位肽830到844的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽830到845的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽829到844的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽829到844的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。例如,P2可以是破伤风毒素的表位肽830到845的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。例如,P2可以是破伤风毒素的表位肽830到844的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。例如,所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
在本申请中,所述P2或其功能活性片段可与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
在本申请中,所述P2或其功能活性片段可与所述RBD或其功能活性片段的N端直接或间接地相连。
在本申请中,所述P2或其功能活性片段可与所述RBD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述RBD或其功能活性片段可与所述P2或其功能活性片段在框内融合。
在本申请中,所述foldon结构域或其功能活性片段可以包含噬菌体T4纤维蛋白C末端的氨基酸残基。
在本申请中,所述foldon结构域或其功能活性片段可以包含噬菌体T4纤维蛋白C末端的27个氨基酸残基或突变体。在本申请中,foldon结构域可以是噬菌体T4纤维蛋白C末端的27个残基截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的截短体或增长体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。
例如,所述foldon结构域或其功能活性片段可以包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
在本申请中,所述foldon结构域或其功能活性片段可以与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
在本申请中,所述foldon结构域或其功能活性片段可以与所述RBD或其功能活性片段的N端直接或间接相连。
在本申请中,所述foldon结构域或其功能活性片段可以与所述RBD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述RBD或其功能活性片段可以与所述foldon结构域或其功能活性片段在框内融合。
在本申请中,所述铁蛋白或其功能活性片段可包含粉纹夜蛾铁蛋白或其功能活性片段。
在本申请中,所述粉纹夜蛾铁蛋白或其功能活性片段可包括粉纹夜蛾铁蛋白的重链或轻链。
在本申请中,铁蛋白可以是粉纹夜蛾或幽门螺旋杆菌铁蛋白的突变体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。在本申请中,“粉纹夜蛾铁蛋白的轻链”、“LC”可以与“ferritin LC”互换使用。在本申请中,“粉纹夜蛾铁蛋白的重链”、“HC”可以与“ferritin HC”互换使用。在本申请中,“幽门螺杆菌铁蛋白”可以与“HP ferritin”互换使用。
在本申请中,所述粉纹夜蛾铁蛋白的重链可包含SEQ ID NO:70所示的氨基酸序列。
在本申请中,所述粉纹夜蛾铁蛋白的轻链可包含SEQ ID NO:71所示的氨基酸序列。
在本申请中,所述铁蛋白或其功能活性片段可包含幽门螺杆菌铁蛋白或其功能活性片段。
在本申请中,所述幽门螺杆菌铁蛋白或其功能活性片段可包含SEQ ID NO:72所示的氨基酸序列。
在本申请中,所述铁蛋白或其功能活性片段可包含SEQ ID NO:70-72中任一项所示的氨基酸序列。
在本申请中,所述铁蛋白或其功能活性片段可以与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
例如,所述铁蛋白或其功能活性片段可以与所述RBD或其功能活性片段的N端直接或间接地相连。
例如,所述铁蛋白或其功能活性片段可以与所述RBD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述RBD或其功能活性片段可以与所述铁蛋白或其功能活性片段在框内 融合。
在本申请中,术语“乙肝表面抗原(HBsAg)”通常是指乙肝病毒最外层包膜中的一种外壳蛋白。例如,乙肝表面抗原的氨基酸序列可以为NCBI中的蛋白序列检索号AAA45524、ANJ76941、CAA24234或AAC34729对应的序列,也可以适宜地截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸,或有蛋白的突变,例如缺失、置换或插入一个或多个氨基酸。
例如,所述乙肝表面抗原(HBsAg)或其功能活性片段可包含SEQ ID NO:73所示的氨基酸序列。
在本申请中,所述乙肝表面抗原(HBsAg)或其功能活性片段可以与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
在本申请中,所述乙肝表面抗原(HBsAg)或其功能活性片段可以与所述RBD或其功能活性片段的N端直接或间接地相连。
在本申请中,所述乙肝表面抗原(HBsAg)或其功能活性片段可以与所述RBD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述RBD或其功能活性片段可以与所述乙肝表面抗原(HBsAg)或其功能活性片段在框内融合。
在本申请中,所述“直接或间接相连”通常是指两段序列直接相连和间接相连这两种不同的连接方式,其中的直接相连是指两段序列之间的连接没有任何人为添加的其余序列,如柔性Linker、刚性Linker或可剪切Linker等的参与。间接相连则是指两段序列是通过人为添加连接序列,例如柔性Linker、刚性Linker或可剪切Linker等手段实现的两段序列的连接方式。在某些实施方式中,所述柔性Linker可以包含GSGSG(SEQ ID NO:89)的氨基酸序列。在某些实施方式中,所述连接序列可以包含SFTVEKGIYQTSNF(SEQ ID NO:90)的氨基酸序列。
在本申请中,所述融合蛋白还可以包含信号肽。在某些实施方式中,可以在所述融合蛋白的N段添加MGWSCIILFLVATATGVHS(SEQ ID NO:91)的信号肽序列。
在本申请中,所述的融合蛋白可包含SEQ ID NO:1-17、80和86中任一项所示的氨基酸序列。
另一方面,本申请提供了一种融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段;以及2)一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原 (HBsAg)或其功能活性片段。
在本申请中,所述NTD或其功能活性片段可以来源于SARS-CoV-2野生型S蛋白的NTD,也可以来源于SARS-CoV-2突变体S蛋白的NTD。在某些实施方式中,所述NTD或其功能活性片段可以在L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246中的一个或多个氨基酸位点处包含氨基酸突变。在某些实施方式中,所述NTD或其功能活性片段可以包含L18F、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I中的一个或多个氨基酸突变。
在本申请中,所述NTD或其功能活性片段可以包含Gamma突变株S蛋白的NTD。在某些实施方式中,所述NTD可以包含L18F、T20N、P26S、D138Y和R190S氨基酸突变。例如,所述NTD可以包含SEQ ID NO:84所示的氨基酸序列。
在本申请中,所述NTD或其功能活性片段可以包含例如Beta突变株S蛋白的NTD。在某些实施方式中,所述NTD或其功能活性片段可以包含L18F、D80A、D215G、L242-244del和R246I突变。在某些实施方式中,所述NTD可以包含SEQ ID NO:77所示的氨基酸序列。
在本申请中,所述P2或其功能活性片段可以包含破伤风毒素的表位肽。
例如,P2可以是破伤风毒素的表位肽830到844的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽830到845的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽829到844的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽829到844的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。例如,P2可以是破伤风毒素的表位肽830到845的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。例如,P2可以是破伤风毒素的表位肽830到844的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。例如,所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
在本申请中,所述P2或其功能活性片段可以与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
在本申请中,所述P2或其功能活性片段可以与所述NTD或其功能活性片段的N端直接或间接地相连。
在本申请中,所述P2或其功能活性片段可以与所述NTD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述NTD或其功能活性片段可以与所述P2或其功能活性片段在框内融合。
在本申请中,所述foldon结构域或其功能活性片段可包含噬菌体T4纤维蛋白C末端的氨基酸残基。在某些实施方式中,所述噬菌体T4纤维蛋白C末端的氨基酸残基可以包含SEQ ID NO:78所示的氨基酸序列。
在本申请中,所述foldon结构域或其功能活性片段可包含噬菌体T4纤维蛋白C末端的27个氨基酸残基或突变体。在本申请中,foldon结构域可以是噬菌体T4纤维蛋白C末端的27个残基截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的截短体或增长体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。
例如,所述foldon结构域或其功能活性片段可包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
在本申请中,所述foldon结构域或其功能活性片段可以与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
在本申请中,所述foldon结构域或其功能活性片段可以与所述NTD或其功能活性片段的N端直接或间接相连。
在本申请中,所述foldon结构域或其功能活性片段可以与所述NTD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述NTD或其功能活性片段可以与所述foldon结构域或其功能活性片段在框内融合。
在本申请中,所述铁蛋白或其功能活性片段可包含粉纹夜蛾铁蛋白或其功能活性片段。
在本申请中,所述粉纹夜蛾铁蛋白或其功能活性片段可包括粉纹夜蛾铁蛋白的重链或轻链。
在本申请中,铁蛋白可以是粉纹夜蛾或幽门螺旋杆菌铁蛋白的突变体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。在本申请中,“粉纹夜蛾铁蛋白的轻链”、“LC”可以与“ferritin LC”互换使用。在本申请中,“粉纹夜蛾铁蛋白的重链”、“HC”可以与“ferritin HC”互换使用。在本申请中,“幽门螺杆菌铁蛋白”可以与“HP ferritin”互换使用。
在本申请中,所述粉纹夜蛾铁蛋白的重链可包含SEQ ID NO:70所示的氨基酸序列。
在本申请中,所述粉纹夜蛾铁蛋白的轻链可包含SEQ ID NO:71所示的氨基酸序列。
在本申请中,所述铁蛋白或其功能活性片段可包含幽门螺杆菌铁蛋白或其功能活性片段。
在本申请中,所述幽门螺杆菌铁蛋白或其功能活性片段可包含SEQ ID NO:72所示的氨基酸序列。
在本申请中,所述铁蛋白或其功能活性片段可包含SEQ ID NO:70-72中任一项所示的氨基酸序列。
在本申请中,所述铁蛋白或其功能活性片段可以与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
例如,所述铁蛋白或其功能活性片段可以与所述NTD或其功能活性片段的N端直接或间接地相连。
例如,所述铁蛋白或其功能活性片段可以与所述NTD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述NTD或其功能活性片段可以与所述铁蛋白或其功能活性片段在框内融合。
在本申请中,术语“乙肝表面抗原(HBsAg)”通常是指乙肝病毒最外层包膜中的一种外壳蛋白。例如,乙肝表面抗原的氨基酸序列可以为NCBI中的蛋白序列检索号AAA45524、ANJ76941、CAA24234或AAC34729对应的序列,也可以适宜地截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸,或有蛋白的突变,例如缺失、置换或插入一个或多个氨基酸。
例如,所述乙肝表面抗原(HBsAg)或其功能活性片段可包含SEQ ID NO:73所示的氨基酸序列。
在本申请中,所述乙肝表面抗原(HBsAg)或其功能活性片段可以与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
在本申请中,所述乙肝表面抗原(HBsAg)或其功能活性片段可以与所述NTD或其功能活性片段的N端直接或间接地相连。
在本申请中,所述乙肝表面抗原(HBsAg)或其功能活性片段可以与所述NTD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述NTD或其功能活性片段可以与所述乙肝表面抗原(HBsAg)或其功能活性片段在框内融合。
在本申请中,所述“直接或间接相连”通常是指两段序列直接相连和间接相连这两种不同的连接方式,其中的直接相连是指两段序列之间的连接没有任何人为添加的其余序列,如柔性Linker、刚性Linker或可剪切Linker等的参与。间接相连则是指两段序列是通过人为添 加连接序列,例如柔性Linker、刚性Linker或可剪切Linker等手段实现的两段序列的连接方式。在某些实施方式中,所述柔性Linker可以包含GSGSG(SEQ ID NO:89)的氨基酸序列。在某些实施方式中,所述连接序列可以包含SFTVEKGIYQTSNF(SEQ ID NO:90)的氨基酸序列。
在本申请中,所述融合蛋白还可以包含信号肽。在某些实施方式中,可以在所述融合蛋白的N段添加MGWSCIILFLVATATGVHS(SEQ ID NO:91)的信号肽序列。
在本申请中,所述的融合蛋白可包含SEQ ID NO:20-36、81和87中任一项所示的氨基酸序列。
另一方面,本申请提供了一种融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及2)SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
在本申请中,所述RBD或其功能活性片段可以来源于SARS-CoV-2野生型S蛋白的RBD,也可以来源于SARS-CoV-2突变体S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以在K417、L452、T478、E484、和N501中的一个或多个氨基酸位点处包含氨基酸突变。
在本申请中,所述RBD或其功能活性片段可以包含Gamma突变株S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以包含K417T、E484K和N501Y中的一个或多个氨基酸突变。例如,所述RBD可以包含SEQ ID NO:83所示的氨基酸序列。在本申请中,所述RBD或其功能活性片段可以包含Beta突变株S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以包含K417N、E484K和N501Y中的一个或多个氨基酸突变。例如,所述RBD可以包含SEQ ID NO:76所示的氨基酸序列。
在本申请中,所述NTD或其功能活性片段可以来源于SARS-CoV-2野生型S蛋白的NTD,也可以来源于SARS-CoV-2突变体S蛋白的NTD。在某些实施方式中,所述NTD或其功能活性片段可以在L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246中的一个或多个氨基酸位点处包含氨基酸突变。在某些实施方式中,所述NTD或其功能活性片段可以包含L18F、T19R、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I中的一个或多个氨基酸突变。
在本申请中,所述NTD或其功能活性片段可以包含Gamma突变株S蛋白的NTD。在某些实施方式中,所述NTD可以包含L18F、T20N、P26S、D138Y和R190S氨基酸突变。例如,所述NTD可以包含SEQ ID NO:84所示的氨基酸序列。
在本申请中,所述NTD或其功能活性片段可以包含Beta突变株S蛋白的NTD。在某些实施方式中,所述NTD或其功能活性片段可以包含L18F、D80A、D215G、L242-244del和R246I突变。例如,所述NTD可以包含SEQ ID NO:77所示的氨基酸序列。
在本申请中,所述RBD或其功能活性片段与所述NTD或其功能活性片段直接或间接地相连。
在本申请中,所述RBD或其功能活性片段的N端与所述NTD或其功能活性片段的C端直接或间接地连接。
在本申请中,所述RBD或其功能活性片段的C端与所述NTD或其功能活性片段的N端直接或间接地连接。
在本申请中,所述RBD或其功能活性片段与所述NTD或其功能活性片段在框内融合。
在本申请中,所述的融合蛋白还可以包括一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
在本申请中,所述P2或其功能活性片段可包含破伤风毒素的表位肽。
例如,P2可以是破伤风毒素的表位肽830到844的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽830到845的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽829到844的肽段或其突变体。例如,P2可以是破伤风毒素的表位肽829到844的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。例如,P2可以是破伤风毒素的表位肽830到845的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。例如,P2可以是破伤风毒素的表位肽830到844的肽段截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的肽段。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。
例如,所述破伤风毒素的表位肽可包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
在本申请中,所述P2或其功能活性片段可以与所述RBD或其功能性片段和/或所述NTD或其功能活性片段直接或间接地相连。
例如,所述RBD或其功能活性片段的N端可以与所述NTD或其功能活性片段的C端直接或间接地连接,且所述P2或其功能活性片段的N端可以与所述RBD或其功能活性片段的C端直接或间接地连接。
在本申请中,所述P2或其功能活性片段可以与所述RBD或其功能活性片段和所述NTD 或其功能活性片段在框内融合。
在本申请中,所述foldon结构域或其功能活性片段可包含噬菌体T4纤维蛋白C末端的氨基酸残基。在某些实施方式中,所述噬菌体T4纤维蛋白C末端的氨基酸残基包含SEQ ID NO:78所示的氨基酸序列。
在本申请中,所述foldon结构域或其功能活性片段可包含噬菌体T4纤维蛋白C末端的27个氨基酸残基或突变体。在本申请中,foldon结构域可以是噬菌体T4纤维蛋白C末端的27个残基截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸得到的截短体或增长体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。
例如,所述foldon结构域或其功能活性片段可包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
在本申请中,所述foldon结构域或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
例如,所述foldon结构域或其功能活性片段的N端可以与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端可以与所述NTD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述foldon结构域或其功能活性片段与所述RBD或其功能活性片段和所述NTD或其功能活性片段在框内融合。
在本申请中,所述foldon结构域或其功能活性片段的N端与所述P2或其功能活性片段的C端直接或间接地相连,所述P2或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活性的C端直接或间接地相连。
在本申请中,所述P2或其功能活性片段、foldon结构域或其功能活性片段、所述RBD或其功能活性片段和所述NTD或其功能活性片段在框内融合。
在本申请中,所述铁蛋白或其功能活性片段包含粉纹夜蛾铁蛋白或其功能活性片段。
在本申请中,所述粉纹夜蛾铁蛋白或其功能活性片段包括粉纹夜蛾铁蛋白的重链或轻链。
在本申请中,铁蛋白可以是粉纹夜蛾或幽门螺旋杆菌铁蛋白的突变体。在本申请中,“突变体”通常是指因含有一个或多个差异(突变)而与参比序列不同的序列。该差异可以是取代、缺失或插入一个或多个氨基酸。在本申请中,“粉纹夜蛾铁蛋白的轻链”、“LC”可以与“ferritin LC”互换使用。在本申请中,“粉纹夜蛾铁蛋白的重链”、“HC”可以与“ferritin HC”互换使用。在本申请中,“幽门螺杆菌铁蛋白”可以与“HP ferritin”互换使用。
在本申请中,所述粉纹夜蛾铁蛋白的重链可包含SEQ ID NO:70所示的氨基酸序列。
在本申请中,所述粉纹夜蛾铁蛋白的轻链可包含SEQ ID NO:71所示的氨基酸序列。
在本申请中,所述铁蛋白或其功能活性片段可包含幽门螺杆菌铁蛋白或其功能活性片段。
在本申请中,所述铁蛋白或其功能活性片段可包含SEQ ID NO:70-72任一项所示的氨基酸序列。
在本申请中,所述铁蛋白或其功能活性片段可以与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
例如,所述铁蛋白或其功能活性片段的N端可以与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端可以与所述NTD或其功能活性片段的C端直接或间接地相连。
例如,所述幽门螺杆菌铁蛋白或其功能活性片段的N端可以与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端可以与所述NTD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述幽门螺杆菌铁蛋白或其功能活性片段可以与所述RBD或其功能活性片段和所述NTD或其功能活性片段在框内融合。
在本申请中,术语“乙肝表面抗原(HBsAg)”通常是指乙肝病毒最外层包膜中的一种外壳蛋白。例如,乙肝表面抗原的氨基酸序列可以为NCBI中的蛋白序列检索号AAA45524、ANJ76941、CAA24234或AAC34729对应的序列,也可以适宜地截短或增加N端或C端1、2、3、4、5、或6个或10个氨基酸,或有蛋白的突变,例如缺失、置换或插入一个或多个氨基酸。
在本申请中,所述乙肝表面抗原或其功能活性片段可包含SEQ ID NO:73所示的氨基酸序列。
在本申请中,所述乙肝表面抗原或其功能活性片段可以与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
在本申请中,所述乙肝表面抗原或其功能活性片段的N端可以与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端可以与所述NTD或其功能活性片段的C端直接或间接地相连。
在本申请中,所述乙肝表面抗原或其功能活性片段与所述RBD或其功能活性片段和所述NTD或其功能活性片段在框内融合。
在本申请中,所述“直接或间接相连”通常是指两段序列直接相连和间接相连这两种不同的连接方式,其中的直接相连是指两段序列之间的连接没有任何人为添加的其余序列,如柔性Linker、刚性Linker或可剪切Linker等的参与。间接相连则是指两段序列是通过人为添加连接序列,例如柔性Linker、刚性Linker或可剪切Linker等手段实现的两段序列的连接方式。在某些实施方式中,所述柔性Linker可以包含GSGSG(SEQ ID NO:89)的氨基酸序列。在某些实施方式中,所述连接序列可以包含SFTVEKGIYQTSNF(SEQ ID NO:90)的氨基酸序列。
在本申请中,所述融合蛋白还可以包含信号肽。在某些实施方式中,可以在所述融合蛋白的N段添加MGWSCIILFLVATATGVHS(SEQ ID NO:91)的信号肽序列。
在本申请中,所述的融合蛋白包含SEQ ID NO:39-44、79、85和96中任一项所示的氨基酸序列。
免疫原性组合物
另一方面,本申请提供了一种免疫原性组合物,其包含所述的融合蛋白。
在本申请中,术语“免疫原性组合物”通常是指一种亚单位组合物。在本申请中,亚单位组合物是在将组分混合形成抗原性组合物之前,其中组分已经被分离并纯化到至少50%、至少60%、70%、80%、90%纯度的组合物。例如,亚单位组合物可以是水溶性蛋白的水溶液。例如,所述亚单位组合物可包含洗涤剂。例如,亚单位组合物可包含非-离子、两性离子或离子洗涤剂。例如,亚单位组合物可包含脂类。在一些情形中,免疫原性组合物可包括RBD或其功能活性片段和NTD或其功能活性片段。在一些情形中,免疫原性组合物可包括RBD或其功能活性片段和包含NTD或其功能活性片段的融合蛋白。在一些情形中,免疫原性组合物可包括包含RBD或其功能活性片段的融合蛋白和NTD或其功能活性片段。在一些情形中,免疫原性组合物可包括包含RBD或其功能活性片段的融合蛋白和包含NTD或其功能活性片段的融合蛋白。在一些情形中,所述免疫原性组合物还可以包含一种或几种人流感病毒血凝素蛋白HA。在本申请中,所述HA可以包含SEQ ID NO:92-95中任一项所述的氨基酸序列。
在某些实施方式中,所述的流感血凝素选自的流感毒株为每一年在WHO官网上公布的流行毒株,例如2021年WHO官网公布的流行毒株指的是A型(H1N1)EPI1661231|HA|A/Wisconsin/588/2019|EPI_ISL_404460,A型(H3N2)EPI1843589|HA|A/Cambodia/e0826360/2020|EPI_ISL_944639,B型EPI1394970|HA|B/Washington/02/2019|EPI_ISL_347829和B型EPI529345|HA|B/PHUKET/3073/2013|EPI_ISL_161843。在一个实施方式中制备预防流感和新冠 病毒疫苗组合物包括上述各型别HA蛋白50ug及突变的NTD-RBD-foldon蛋白50μg及疫苗组合中常用的佐剂,例如AS03佐剂。
例如,所述免疫原性组合物可以包含第一组分和第二组分,所述第一组分可以包含本申请所述的RBD或其功能活性片段及NTD或其功能活性片段的融合蛋白,所述融合蛋白还可以包含一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段;所述第二组分可以包含所述RBD或其功能活性片段,所述NTD或其功能活性片段,或任选地一种或几种人流感病毒血凝素蛋白HA。
在本申请中,所述RBD或其功能活性片段可以来源于SARS-CoV-2野生型S蛋白的RBD,也可以来源于SARS-CoV-2突变体S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以在K417、L452、T478、E484和N501中的一个或多个氨基酸位点处包含氨基酸突变。
在本申请中,所述RBD或其功能活性片段可以包含Gamma突变株S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以包含K417T、E484K和N501Y中的一个或多个氨基酸突变。例如,所述RBD可以包含SEQ ID NO:83所示的氨基酸序列。在本申请中,所述RBD或其功能活性片段可以包含Beta突变株S蛋白的RBD。在某些实施方式中,所述RBD或其功能活性片段可以包含K417N、E484K和N501Y中的一个或多个氨基酸突变。在某些实施方式中,所述RBD可以包含SEQ ID NO:76所示的氨基酸序列。
在本申请中,所述NTD或其功能活性片段可以来源于SARS-CoV-2野生型S蛋白的NTD,也可以来源于SARS-CoV-2突变体S蛋白的NTD。在某些实施方式中,所述NTD或其功能活性片段可以在L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246中的一个或多个氨基酸位点处包含氨基酸突变。在某些实施方式中,所述NTD或其功能活性片段可以包含L18F、T19R、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I中的一个或多个氨基酸突变。
在本申请中,所述NTD或其功能活性片段可以包含Gamma突变株S蛋白的NTD。在某些实施方式中,所述NTD可以包含L18F、T20N、P26S、D138Y和R190S氨基酸突变。例如,所述NTD可以包含SEQ ID NO:84所示的氨基酸序列。
在本申请中,所述NTD或其功能活性片段可以包含Beta突变株S蛋白的NTD。在某些实施方式中,所述NTD或其功能活性片段可以包含L18F、D80A、D215G、L242-244del和R246I突变。例如,所述NTD可以包含SEQ ID NO:77所示的氨基酸序列。
在本申请中,所述免疫原性组合物还可包括佐剂。例如,所述的佐剂可以包括铝盐(例如氢氧化铝凝胶(alum)或磷酸铝),但也可以是钙盐、铁盐或锌盐,或者可以是酰化酪氨酸或酰化糖、阳离子或阴离子衍生化多糖或聚磷腈的不溶性悬浮液。例如,所述的免疫原性组合物还可以选择为Th1型应答优先诱导物。例如,Th1型应答优先诱导物可以包括单磷酰脂质A或其衍生物。例如,所述佐剂可以是单磷酰脂质A(例如,3-脱-O-酰化单磷酰脂质A(3D-MPL))和铝盐的组合。一种佐剂增强系统可以包括单磷酰脂质A和皂苷衍生物的组合,特别是WO94/00153公开的QS21和3D-MPL的组合,或者如WO96/33739公开的用胆固醇将QS21猝灭从而使反应原性较弱的一种组合物。例如,所述佐剂还可以是WO95/17210中所述的佐剂,其在水包油乳剂中含有QS21、3D-MPL和生育酚。例如,所述佐剂可以是吐温80、山梨糖醇三油酸酯和角鲨烯混合后于高压条件下进行微流化形成的均一小滴状乳液。例如,所述佐剂可包含寡核苷酸的未甲基化CpG(WO96/02555)。
在本申请中,所述的免疫原性组合物包含第一组分,所述第一组分包含所述的融合蛋白;以及第二组分,所述第二组分包含所述的融合蛋白。
在本申请中,所述的免疫原性组合物,其可按下述重量比配制而成:1)第一组分(1-15重量份);和/或2)第二组分(1-15重量份)。
在本申请中,所述的免疫原性组合物,其可包含:1)5-60μg第一组分;和/或2)5-60μg第二组分。
在本申请中,术语“重量份”通常是指免疫原性组合物的第一组分和第二组分的重量比。在本申请中,免疫原性组合物每剂量可含有如下成份:第一组分5-60μg和第二组分5-60μg。例如,免疫原性组合物可以包含5μg、10μg、20μg、30μg或40μg的第一组分。例如,免疫原性组合物可以包含5μg、10μg、20μg、30μg或40μg的第二组分。
另一方面,本申请提供了一种免疫原性组合物,其包含第一组分,所述第一组分包含SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及第二组分,所述第二组分包含SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
在本申请中,所述的免疫原性组合物,其可按下述重量比配制而成:1)第一组分(1-15重量份);和/或2)第二组分(1-15重量份)。
在本申请中,所述的免疫原性组合物,其可包含:1)5-60μg第一组分;和/或2)5-60μg第二组分。
在本申请中,所述RBD可包含SEQ ID NO:18-19中任一项所示的氨基酸序列。
在本申请中,所述NTD包含SEQ ID NO:37-38中任一项所示的氨基酸序列。
在本申请中,术语“重量份”通常是指免疫原性组合物的第一组分和第二组分的重量比。在本申请中,免疫原性组合物每剂量可含有如下成份:第一组分5-60μg和第二组分5-60μg。例如,免疫原性组合物可以包含5μg、10μg、20μg、30μg或40μg的第一组分。例如,免疫原性组合物可以包含5μg、10μg、20μg、30μg或40μg的第二组分。
在一些情形中,所述免疫原性组合物可以包含第一组分NTD-RBD-foldon蛋白20~100μg;以及第二组分NTD-foldon蛋白20~100μg,或RBD-foldon蛋白20~100μg,或流感病毒血凝素蛋白15~80μg。
在某些实施方式中,所述的免疫原性组合物包含NTD-RBD-foldon(Gamma突变株)蛋白20~100μg,NTD-foldon(Gamma突变株)蛋白20~100μg,以及冻干保护剂,所述的冻干保护剂按照1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4)的配方配制。
在某些实施方式中,所述的免疫原性组合物包含NTD-RBD-foldon(Gamma突变株)蛋白20~100μg,RBD-foldon(Gamma突变株)蛋白20~100μg,以及冻干保护剂,所述的冻干保护剂按照1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4)的配方配制。
在某些实施方式中,所述的免疫原性组合物包含NTD-RBD-foldon(Gamma突变株)蛋白20~100μg,流感病毒血凝素蛋白15~180μg,以及冻干保护剂,所述的冻干保护剂按照1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4)的配方配制。
另一方面,本发明提供了一种疫苗,其包含所述的融合蛋白,或所述的免疫原性组合物,及任选地药学上可接受的佐剂。
在某些实施方式中,所述疫苗包含0.5mL的融合蛋白或免疫原性组合物,及0.5mL任选地药学上可接受的佐剂。
本发明所述的佐剂选自角鲨烯、MF59、AS03、单磷脂酰脂质A、鞭毛蛋白、CpG-ODN、胞壁酰二肽、以及铝或钙盐的小分子中的一种或多种。这些佐剂均是本领域所熟知并可通过若干商业渠道获得的。
在某些实施方式中,所述佐剂是氢氧化铝佐剂。
在某些实施方式中,所述佐剂是MF59佐剂。
在某些实施方式中,所述佐剂优选地是AS03佐剂。
所述AS03佐剂的成分包括10.69mg的角鲨烯、11.86mg的α-生育酚、4.86mg的聚山梨酯80、3.53mg的氯化钠、0.09mg的氯化钾、0.51mg的磷酸氢二钠、0.09mg的磷酸二氢钾。
所述MF59佐剂的成分包括4.5%的角鲨烯,0.5%Tween 80,0.5%Span85。
在某些实施方式中,所述疫苗包含NTD-RBD-foldon蛋白20~100μg,1%(w/v)蔗糖,2%(w/v)甘氨酸,0.02%(w/v)吐温80和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),以及AS03佐剂。
在某些实施方式中,所述疫苗包含NTD-RBD-foldon蛋白20~100μg,H1N1毒株的HA 15~45μg,H3N2毒株的HA 15~45μg,和B/Washington/02/2019毒株的HA 15~45μg,1%(w/v)蔗糖,2%(w/v)甘氨酸,0.02%(w/v)吐温80和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),以及AS03佐剂。
在某些实施方式中,所述疫苗包含NTD-RBD-foldon蛋白20~100μg,H1N1毒株的HA 15~45μg,H3N2毒株的HA 15~45μg,B/Washington/02/2019毒株的HA 15~45μg,和B/PHUKET/3073/2013毒株的HA 15~45μg,1%(w/v)蔗糖,2%(w/v)甘氨酸,0.02%(w/v)吐温80和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),以及AS03佐剂。
在某些实施方式中,所述疫苗包含NTD-RBD-foldon蛋白20~100μg,H1N1毒株的HA 15~45μg,H3N2毒株的HA 15~45μg,和B/Washington/02/2019毒株的HA 15~45μg,1%(w/v)蔗糖,2%(w/v)甘氨酸,0.02%(w/v)吐温80和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),以及MF59佐剂。
在某些实施方式中,所述疫苗包含NTD-RBD-foldon蛋白20~100μg,H1N1毒株的HA 15~45μg,H3N2毒株的HA 15~45μg,B/Washington/02/2019毒株的HA 15~45μg,和B/PHUKET/3073/2013毒株的HA 15~45μg,1%(w/v)蔗糖,2%(w/v)甘氨酸,0.02%(w/v)吐温80和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),以及MF59佐剂。
在某些优选的实施方式中,所述疫苗包含NTD-RBD-foldon蛋白50μg,H1N1毒株的HA 45μg,H3N2毒株的HA 45μg,B/Washington/02/2019毒株的HA 45μg,和B/PHUKET/3073/2013毒株的HA 45μg,1%(w/v)蔗糖,2%(w/v)甘氨酸,0.02%(w/v)吐温80和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),以及AS03佐剂。
在某些优选的实施方式中,所述疫苗包含NTD-RBD-foldon蛋白50μg,H1N1毒株的HA 45μg,H3N2毒株的HA 45μg,B/Washington/02/2019毒株的HA 45μg,和B/PHUKET/3073/2013毒株的HA 45μg,1%(w/v)蔗糖,2%(w/v)甘氨酸,0.02%(w/v)吐温80和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),以及MF59佐剂。
在本申请中,所述RBD-NTD-foldon(Beta突变株)可以包含SEQ ID NO:79所示的氨基酸序列。在本申请中,所述RBD-NTD-foldon(Gamma突变株)可以包含SEQ ID NO:85所示的氨基酸序列。在本申请中,所述RBD-NTD-foldon(WIV04-1)可以包含SEQ ID NO:96所示的氨基酸序列。
在本申请中,所述免疫原性组合物还可以包含一种或几种人流感病毒血凝素蛋白HA。在本申请中,所述HA可以包含SEQ ID NO:92-95中任一项所述的氨基酸序列。
在本申请中,所述RBD还可以包含SEQ ID NO:97-108中任一项所示的氨基酸序列。
在本申请中,所述NTD还可以包含SEQ ID NO:109-111中任一项所示的氨基酸序列。
核酸分子、药物组合物、应用
另一方面,本申请还提供了一种或多种分离的核酸分子,其可以编码本申请所述的融合蛋白。在某些实施方式中,所述核酸分子可以包含mRNA。
另一方面,本申请提供了一种药物组合物,其包含所述的融合蛋白,或所述的免疫原性组合物,及任选地药学上可接受的赋形剂。
所述药学上可接受的载体可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在本申请中,所述药物组合物可被配制用于口服给药,静脉内给药,肌肉内给药,在肿瘤部位的原位给药,吸入,直肠给药,阴道给药,经皮给药或通过皮下储存库给药。
所述药物组合物可以用于抑制或延缓疾病或病症的发展或进展,和/或可以减轻和/或稳定疾病或病症的状态。
本申请所述的药物组合物可以包含预防和/或治疗有效量的所述融合蛋白、或所述的免疫原性组合物。
所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的疾病或病症和/或其任何并发症而所需的剂量。
在本申请中,术语“有效量”通常是指可以缓解或者消除受试者的疾病或症状,或者可以预防性地抑制或防止疾病或症状发生的药物的量。通常,可根据受试者的体重、年龄、性别、饮食、排泄速率、过往病史、现用治疗、给药时间、剂型、给药方法、给药途径、药物组合、所述受试者的健康状况和交叉感染的潜力、过敏、超敏和副作用、和/或上皮(或内皮)组织疾病发展的程度等来确定具体的有效量。本领域技术人员(例如,医生或兽医)可根据这些或其它条件或要求按比例降低或升高剂量。
在本申请中,所述受试者可以包括人或非人动物。例如,所述非人动物可以选自下组: 猴、鸡、鹅、猫、狗、小鼠和大鼠。此外,非人动物也可以包括任何除人以外的动物物种,例如家畜动物,或啮齿类动物,或灵长类动物,或家养动物,或家禽动物。所述人可以是高加索人、非洲人、亚洲人、闪族人,或其他种族,或各种种族的杂合体。又例如,所述人可以是老年、成年、青少年、儿童或者婴儿。
可以根据在实验动物中的有效量推测在人类中的有效量。例如,Freireich等人描述了动物和人的剂量的相互关系(基于每平方米身体表面的毫克数)(Freireich et al.,Cancer Chemother.Rep.50,219(1966))。身体表面积可以从患者的身高和体重近似确定。参见例如Scientific Tables,Geigy Pharmaceuticals,Ardsley,N.Y.,537(1970)。
另一方面,本申请提供了一种所述的融合蛋白或所述的免疫原性组合物在制备疫苗中的用途。
在本申请中,所述疫苗可用于预防和/或治疗COVID-19。
另一方面,本申请提供了一种所述的融合蛋白或所述的免疫原性组合物,用于治疗和/或预防COVID-19。
另一方面,本申请提供了一种制备COVID-19亚单位疫苗的方法,其包括:
1)提供所述的融合蛋白或所述的免疫原性组合物;以及
2)使1)中所述融合蛋白或免疫原性组合物与药学上可接受的佐剂混合。
另一方面,本申请提供了一种检测SARS-CoV-2中和抗体的方法,其包括:
1)向受试者施用所述的COVID-19亚单位疫苗;以及
2)检测1)中所述受试者在接受所述COVID-19亚单位疫苗后体内产生的中和抗体。
另一方面,本申请提供了一种治疗和/或预防COVID-19的方法,其包括向受试者施用所述的融合蛋白或所述的免疫原性组合物或所述的COVID-19亚单位疫苗。
本申请还涉及以下的具体实施方式:
1.融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及2)一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
2.根据实施方式1所述的融合蛋白,其中所述RBD源自SARS-CoV-2野生型或其突变体。
3.根据实施方式2所述的融合蛋白,其中所述SAR-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
4.根据实施方式1-3中任一项所述的融合蛋白,其中所述RBD与野生型SARS-CoV-2的 RBD相比,在选自下组的一个或多个氨基酸位点有突变:K417、L452、T478、E484和N501。
5根据实施方式1-4中任一项所述的融合蛋白,其中所述RBD包含氨基酸突变K417T/N、L452R、T478K、E484K和N501Y的一种或多种。
6.根据实施方式1-5中任一项所述的融合蛋白,其中所述RBD包含K417T、E484K和N501Y氨基酸突变。
7.根据实施方式1-6中任一项所述的融合蛋白,其中所述RBD包含K417N、E484K和N501Y氨基酸突变。
8.根据实施方式1-7中任一项所述的融合蛋白,其中所述RBD包含SEQ ID NO:18、19、76、83、97-108中任一项所示的氨基酸序列。
9.根据实施方式1-8任一项所述的融合蛋白,其中所述P2或其功能活性片段包含破伤风毒素的表位肽。
10.根据实施方式9所述的融合蛋白,其中所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
11.根据实施方式1-10任一项所述的融合蛋白,其中所述P2或其功能活性片段与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
12.根据实施方式1-11任一项所述的融合蛋白,其中所述RBD或其功能活性片段与所述P2或其功能活性片段在框内融合。
13.根据实施方式1-12任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段包含噬菌体T4纤维蛋白C末端的氨基酸残基。
14.根据实施方式1-13任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
15.根据实施方式1-14任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
16.根据实施方式1-15任一项所述的融合蛋白,其中所述RBD或其功能活性片段与所述foldon结构域或其功能活性片段在框内融合。
17.根据实施方式1-16任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含粉纹夜蛾铁蛋白或其功能活性片段。
18.根据实施方式17所述的融合蛋白,其中所述粉纹夜蛾铁蛋白或其功能活性片段包括粉纹夜蛾铁蛋白的重链或轻链。
19.根据实施方式18所述的融合蛋白,其中所述粉纹夜蛾铁蛋白的重链包含SEQ ID NO:70所示的氨基酸序列。
20.根据实施方式18-19任一项所述的融合蛋白,其中所述粉纹夜蛾铁蛋白的轻链包含SEQ ID NO:71所示的氨基酸序列。
21.根据实施方式1-20任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含幽门螺杆菌铁蛋白或其功能活性片段。
22.根据实施方式1-21任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含SEQ ID NO:70-72中任一项所示的氨基酸序列。
23.根据实施方式1-22任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
24.根据实施方式1-23任一项所述的融合蛋白,其中所述RBD或其功能活性片段与所述铁蛋白或其功能活性片段在框内融合。
25.根据实施方式1-24任一项所述的融合蛋白,其中所述乙肝表面抗原或其功能活性片段包含SEQ ID NO:73所示的氨基酸序列。
26.根据实施方式1-25任一项所述的融合蛋白,其中所述乙肝表面抗原或其功能活性片段与所述RBD或其功能活性片段的N端或C端直接或间接地相连。
27.根据实施方式1-26任一项所述的融合蛋白,其中所述RBD或其功能活性片段与所述乙肝表面抗原或其功能活性片段在框内融合。
28根据实施方式11-27任一项所述的融合蛋白,其中所述直接或间接相连可以包含通过连接子相连。
29根据实施方式28所述的融合蛋白,其中所述连接子可以包含刚性接头,柔性接头或其他序列。
30根据实施方式28-29任一项所述的融合蛋白,其中所述连接子可以包含SEQ ID NO:89-90中任一项所示的氨基酸序列。
31.根据实施方式1-30任一项所述的融合蛋白,其包含SEQ ID NO:1-17、80和86中任一项所示的氨基酸序列。
32.融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段;以及2)一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
33.根据实施方式32所述的融合蛋白,其中所述NTD源自SARS-CoV-2野生型或其突变体 S蛋白的NTD。
34.根据实施方式33所述的融合蛋白,其中所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
35.根据实施方式32-34中任一项所述的融合蛋白,其中所述NTD包含在选自下组的一个或多个氨基酸位点的突变:L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246。
36.根据实施方式32-35中任一项所述的融合蛋白,其中所述NTD包含选自下组的一个或多个氨基酸突变:L18F、T19R、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I。
37根据实施方式32-36中任一项所述的融合蛋白,其中所述NTD包含L18F、D80A、D215G、L242-244del和R246I突变。
38根据实施方式32-37中任一项所述的融合蛋白,其中所述NTD包含L18F、D80A、D215G、L242-244del和R190S氨基酸突变。
39.根据实施方式32-38中任一项所述的融合蛋白,其中所述NTD包含L18F、T20N、P26S、D138Y和R246I氨基酸突变。
40.根据实施方式32-39中任一项所述的融合蛋白,其中所述NTD包含SEQ ID NO:37、38、77、84和109-111中任一项所示的氨基酸序列。
41.根据实施方式32-40任一项所述的融合蛋白,其中所述P2或其功能活性片段包含破伤风毒素的表位肽。
42.根据实施方式41所述的融合蛋白,其中所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
43.根据实施方式32-42任一项所述的融合蛋白,其中所述P2或其功能活性片段与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
44.根据实施方式32-43任一项所述的融合蛋白,其中所述NTD或其功能活性片段与所述P2或其功能活性片段在框内融合。
45.根据实施方式32-44任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段包含噬菌体T4纤维蛋白C末端的氨基酸残基。
46.根据实施方式32-45任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
47.根据实施方式32-46任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段 与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
48.根据实施方式32-47任一项所述的融合蛋白,其中所述NTD或其功能活性片段与所述foldon结构域或其功能活性片段在框内融合。
49.根据实施方式32-48任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含粉纹夜蛾铁蛋白或其功能活性片段。
50.根据实施方式49所述的融合蛋白,其中所述粉纹夜蛾铁蛋白或其功能活性片段包括粉纹夜蛾铁蛋白的重链或轻链。
51.根据实施方式50所述的融合蛋白,其中所述粉纹夜蛾铁蛋白的重链包含SEQ ID NO:70所示的氨基酸序列。
52.根据实施方式50-51任一项所述的融合蛋白,其中所述粉纹夜蛾铁蛋白的轻链包含SEQ ID NO:71所示的氨基酸序列。
53.根据实施方式32-52任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含幽门螺杆菌铁蛋白或其功能活性片段。
54.根据实施方式32-53任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含SEQ ID NO:70-72任一项所示的氨基酸序列。
55.根据实施方式32-54任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
56.根据实施方式32-55任一项所述的融合蛋白,其中所述NTD或其功能活性片段与所述铁蛋白或其功能活性片段在框内融合。
57.根据实施方式32-56任一项所述的融合蛋白,其中所述乙肝表面抗原或其功能活性片段包含SEQ ID NO:73所示的氨基酸序列。
58.根据实施方式32-57任一项所述的融合蛋白,其中所述乙肝表面抗原或其功能活性片段与所述NTD或其功能活性片段的N端或C端直接或间接地相连。
59.根据实施方式32-58任一项所述的融合蛋白,其中所述NTD或其功能活性片段与所述乙肝表面抗原或其功能活性片段在框内融合。
60根据实施方式43-59任一项所述的融合蛋白,其中所述直接或间接相连可以包含通过连接子相连。
61.根据实施方式60所述的融合蛋白,其中所述连接子可以包含刚性接头,柔性接头或其他序列。
62.根据实施方式60-61任一项所述的融合蛋白,其中所述连接子可以包含SEQ ID NO:89- 90中任一项所示的氨基酸序列。
63.根据实施方式32-62中任一项所述的融合蛋白,其包含SEQ ID NO:20-36、81和87中任一项所示的氨基酸序列。
64.融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及2)SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
65.根据实施方式64所述的融合蛋白,其中所述RBD源自SARS-CoV-2野生型或其突变体。
66.根据实施方式65所述的融合蛋白,其中所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
67.根据实施方式64-66中任一项所述的融合蛋白,其中所述RBD在选自下组的一个或多个氨基酸位点有突变:K417、L452、T478、E484和N501。
68.根据实施方式64-67中任一项所述的融合蛋白,其中所述RBD包含氨基酸突变K417T/N、L452R、T478K、E484K和N501Y的一种或多种。
69.根据实施方式64-68中任一项所述的融合蛋白,其中所述RBD包含K417T、E484K和N501Y氨基酸突变。
70.根据实施方式64-69中任一项所述的融合蛋白,其中所述RBD包含K417N、E484K和N501Y氨基酸突变。
71.根据实施方式64-70中任一项所述的融合蛋白,其中所述RBD包含SEQ ID NO:18、19、76、83、97-108中任一项所示的氨基酸序列。
72.根据实施方式64-71任一项所述的融合蛋白,其中所述NTD源自SARS-CoV-2野生型或其突变体。
73.根据实施方式72所述的融合蛋白,其中所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
74.根据实施方式64-73中任一项所述的融合蛋白,其中所述NTD包含在选自下组的一个或多个氨基酸位点的突变:L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246。
75根据实施方式64-74中任一项所述的融合蛋白,其中所述NTD包含选自下组的一个或多个氨基酸突变:L18F、T19R、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I。
76根据实施方式64-75中任一项所述的融合蛋白,其中所述NTD包含L18F、D80A、 D215G、L242-244del和R246I突变。
77根据实施方式64-76中任一项所述的融合蛋白,其中所述NTD包含L18F、D80A、D215G、L242-244del和R190S氨基酸突变。
78.根据实施方式64-77中任一项所述的融合蛋白,其中所述NTD包含L18F、T20N、P26S、D138Y和R246I氨基酸突变。
79.根据实施方式64-78中任一项所述的融合蛋白,其中所述NTD包含SEQ ID NO:37、38、77、84和109-111中任一项所示的氨基酸序列。
80.根据实施方式64-79任一项所述的融合蛋白,其还包括一种或多种选自下组的多肽:P2或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
81.根据实施方式64-80任一项所述的融合蛋白,其中所述RBD或其功能活性片段与所述NTD或其功能活性片段直接或间接地相连。
82.根据实施方式64-81任一项所述的融合蛋白,其中所述RBD或其功能活性片段与所述NTD或其功能活性片段在框内融合。
83.根据实施方式64-82任一项所述的融合蛋白,其中所述P2或其功能活性片段包含破伤风毒素的表位肽。
84.根据实施方式83所述的融合蛋白,其中所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
85.根据实施方式64-84任一项所述的融合蛋白,其中所述P2或其功能活性片段与所述RBD或其功能性片段和/或所述NTD或其功能活性片段直接或间接地相连。
86.根据实施方式64-85任一项所述的融合蛋白,其中所述P2或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
87.根据实施方式64-86任一项所述的融合蛋白,其中所述P2或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性片段的N端与所述NTD或其功能活性片段的C端直接或间接地相连。
88.根据实施方式64-87任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段包含噬菌体T4纤维蛋白C末端的氨基酸残基。
89.根据实施方式64-88任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
90.根据实施方式64-89任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段 与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
91.根据实施方式64-90任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
92.根据实施方式64-91任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活性片段的C端直接或间接地相连。
93.根据实施方式64-92任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段的N端与所述P2或其功能活性片段的C端直接或间接地相连,所述P2或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活性的C端直接或间接地相连。
94.根据实施方式64-93任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含粉纹夜蛾铁蛋白或其功能活性片段。
95.根据实施方式94所述的融合蛋白,其中所述粉纹夜蛾铁蛋白或其功能活性片段包括粉纹夜蛾铁蛋白的重链或轻链。
96.根据实施方式95所述的融合蛋白,其中所述粉纹夜蛾铁蛋白的重链包含SEQ ID NO:70所示的氨基酸序列。
97.根据实施方式95-96任一项所述的融合蛋白,其中所述粉纹夜蛾铁蛋白的轻链包含SEQ ID NO:71所示的氨基酸序列。
98.根据实施方式64-97任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含幽门螺杆菌铁蛋白或其功能活性片段。
99.根据实施方式64-98任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含SEQ ID NO:70-72任一项所示的氨基酸序列。
100.根据实施方式64-99任一项所述地融合蛋白,其中所述铁蛋白或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
101.根据实施方式64-100任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
102.根据实施方式64-101任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活性片段的C端直接或间接地相连。
103.根据实施方式64-102任一项所述的融合蛋白,其中所述乙肝表面抗原或其功能活性片段 包含SEQ ID NO:73所示的氨基酸序列。
104.根据实施方式64-103任一项所述的融合蛋白,其中所述乙肝表面抗原或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段直接或间接地相连。
105.根据实施方式64-104任一项所述的融合蛋白,其中所述乙肝表面抗原或其功能活性片段与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
106.根据实施方式81-105任一项所述的融合蛋白,其中所述直接或间接相连可以包含通过连接子相连。
107.根据实施方式106所述的融合蛋白,其中所述连接子可以包含刚性接头,柔性接头或其他序列。
108.根据实施方式81-107所述的融合蛋白,其中所述连接子可以包含SEQ ID NO:89-90中任一项所示的氨基酸序列。
109.根据实施方式64-108中任一项所述的融合蛋白,其包含SEQ ID NO:39-44、79、85和96中任一项所示的氨基酸序列。
110.免疫原性组合物,其包含实施方式1-109中任一项所述的融合蛋白。
111.根据实施方式110所述的免疫原性组合物,其包含第一组分,所述第一组分包含SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及第二组分,所述第二组分包含实施方式1-109中任一项所述的融合蛋白。
112.根据实施方式110所述的免疫原性组合物,其包含第一组分,所述第一组分包含实施方式1-109中任一项所述的融合蛋白;以及第二组分,所述第二组分包含SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
113.根据实施方式110所述的免疫原性组合物,其包含第一组分,所述第一组分包含实施方式1-109中任一项所述的融合蛋白;以及第二组分,所述第二组分包含实施方式1-109中任一项所述的融合蛋白。
114.根据实施方式110-113任一项所述的免疫原性组合物,其按下述重量比配制而成:1)第一组分(1-15重量份);和/或2)第二组分(1-15重量份)。
115.根据实施方式110-114任一项所述的免疫原性组合物,其包含:1)5-60μg第一组分;和/或2)5-60μg第二组分。
116.免疫原性组合物,其包含第一组分,所述第一组分包含SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及第二组分,所述第二组分包含SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
117.根据实施方式116所述的免疫原性组合物,其中所述RBD源自SARS-CoV-2野生型或其突变体。
118.根据实施方式117所述的免疫原性组合物,其中所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
119.根据实施方式116-118中任一项所述的免疫原性组合物,其中所述RBD在选自下组的一个或多个氨基酸位点有突变:K417、L452、T478、E484和N501。
120.根据实施方式116-119中任一项所述的免疫原性组合物,其中所述RBD包含氨基酸突变K417T/N、L452R、T478K、E484K和N501Y的一种或多种。
121.根据实施方式116-120中任一项所述的免疫原性组合物,其中所述RBD包含K417T、E484K和N501Y氨基酸突变。
122.根据实施方式116-121中任一项所述的免疫原性组合物,其中所述RBD包含K417N、E484K和N501Y氨基酸突变。
123.根据实施方式116-122中任一项所述的免疫原性组合物,其中所述RBD包含SEQ ID NO:18、19、76、83、97-108中任一项所示的氨基酸序列。
124.根据实施方式116-123任一项所述的免疫原性组合物,其中所述NTD源自SARS-CoV-2野生型或其突变体。
125.根据实施方式124所述的免疫原性组合物,其中所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Delta突变体和Alpha突变体。
126.根据实施方式116-125中任一项所述的免疫原性组合物,其中所述NTD包含在选自下组的一个或多个氨基酸位点的突变:L18、T20、P26、D80、D138、R190、D215、L242-244和R246。
127.根据实施方式116-126中任一项所述的免疫原性组合物,其中所述NTD包含选自下组的一个或多个氨基酸突变:L18F、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I。
128.根据实施方式116-127中任一项所述的免疫原性组合物,其中所述NTD包含L18F、D80A、D215G、L242-244del和R246I突变。
129.根据实施方式116-128中任一项所述的免疫原性组合物,其中所述NTD包含L18F、D80A、D215G、L242-244del和R190S氨基酸突变。
130.根据实施方式116-129中任一项所述的免疫原性组合物,其中所述NTD包含L18F、T20N、P26S、D138Y和R246I氨基酸突变。
131.根据实施方式116-130中任一项所述的免疫原性组合物,其中所述NTD包含SEQ ID NO:37、38、77、84和109-111中任一项所示的氨基酸序列。
132.根据实施方式116-131任一项所述的免疫原性组合物,其按下述重量比配制而成:1)第一组分(1-15重量份);和/或2)第二组分(1-15重量份)。
133.根据实施方式111-132任一项所述的免疫原性组合物,其包含:1)5-60μg第一组分;和/或2)5-60μg第二组分。
134.根据实施方式116-133任一项所述的免疫原性组合物,其中所述RBD包含SEQ ID NO:18-19中任一项所示的氨基酸序列。
135.根据实施方式116-134任一项所述的免疫原性组合物,其中所述NTD包含SEQ ID NO:37-38中任一项所示的氨基酸序列。
136.一种或多种分离的核酸分子,其编码权利要求1-109中任一项所述的融合蛋白。
137.根据权利要求136所述的核酸分子,其包含mRNA。
138.药物组合物,其包含实施方式1-109任一项所述的融合蛋白,或实施方式110-135中任一项所述的免疫原性组合物,或实施方式136-137中任一项所述的核酸分子,及任选地药学上可接受的赋形剂。
139.实施方式1-109任一项所述的融合蛋白或实施方式110-135任一项所述的免疫原性组合物在制备疫苗中的用途。
140.根据实施方式139所述的用途,其中所述疫苗用于预防和/或治疗COVID-19。
141.实施方式1-109任一项所述的融合蛋白,或实施方式110-135任一项所述的免疫原性组合物,或实施方式136-137中任一项所述的核酸分子,用于治疗和/或预防COVID-19。
142.一种制备COVID-19亚单位疫苗的方法,其包括:
1)提供实施方式1-109任一项所述的融合蛋白或实施方式110-135任一项所述的免疫原性组合物;以及
2)使1)中所述融合蛋白或免疫原性组合物与药学上可接受的佐剂混合。
143.一种检测SARS-CoV-2中和抗体的方法,其包括:
1)向受试者施用实施方式142所述的COVID-19亚单位疫苗;以及
2)检测1)中所述受试者在接受所述COVID-19亚单位疫苗后体内产生的中和抗体。
144.一种治疗和/或预防COVID-19的方法,其包括向受试者施用实施方式1-109任一项所述的融合蛋白,或实施方式110-135任一项所述的免疫原性组合物,或实施方式136-137中任一项所述的核酸分子,或实施方式142所述的COVID-19亚单位疫苗。
不欲被任何理论所限,下文中的实施例仅仅是为了阐释本申请的融合蛋白、免疫原性组合物、制备方法和用途等,而不用于限制本申请发明的范围。此外,实施例中无论是否有6个或8个HIS标签或N端的信号肽,不影响保护范围,熟悉本领域的技术人员根据本申请发明的内容对实施例进行非本质的改进和调整,仍属于本申请发明的保护范围。
实施例
实施例1构建RBD-P2-6*HIS(GP101150-7)重组表达质粒
信号肽添加:MGVPAVPEASSPRWGTLLLAIFLAASRGLVAA(SEQ ID NO:74)。载体选择:pcdna3.1(+),按照宿主CHO细胞进行密码子优化。
片段的克隆。RBD-P2-6*HIS的引物通过PCR的方法扩增得到片段PCR产物,通过多段重组的方法重组到目的载体pcdna3.1(+)(BamHI-XhoI酶切载体)中,得到RBD-P2-6*HIS(GP101150-7)重组表达质粒。反应体系如表1所示。
表1处理好的目的片段与载体连接反应体系
名称 | 体积 |
酶切后载体 | 5μl |
纯化的PCR产物 | 5μl |
无缝组装MIX | 10μl |
总体积 | 20μl |
以上连接液在52℃恒温环境下连接30min,得到RBD-P2-6*HIS(GP101150-7)重组表达质粒。
转化的方法:(1)将浓度在100ng/μl的RBD-P2-6*HIS(GP101150-7)重组表达质粒吸取1-3μl加入到100μl的感受态细胞里,轻轻晃动旋转以混匀,冰上放置3分钟。(2)42℃水浴90s不要摇动。(3)冰浴中放置3分钟左右。(4)每管中加入500-800μl 37℃预温LB培养基,37℃摇床200rpm温和振荡40分钟。
RBD-P2-6*HIS(GP101150-7)重组表达质粒的验证:(1)制备含相应抗性的琼脂平板。(2)取100μl菌液,然后平铺在含有相应抗性的琼脂板,用无菌玻璃涂布器轻轻将细菌涂在平板表面,将平板置于37℃培养15分钟。(3)倒置平板于37℃培养12-16小时可出现菌落。(4)平板挑菌,37℃250转/分钟摇菌14小时,用菌液进行PCR鉴定,将阳性克隆菌液送测序。
克隆质粒的鉴定方法:PCR扩增RBD-P2-6*HIS片段,鉴定引物序列由公司内部引物部合成预期扩增的片段长度为861bp,PCR反应采用20μL体系:引物0.5μL,模板菌液2μL,聚合酶缓冲液0.5μL,buffer 3μl,ddH
2O 14μL。循环参数:96℃预变性3min;95℃15S,58℃15S,72℃20s,23个循环,72℃终延伸1min。以菌液PCR方法筛选阳性克隆,获得的阳性菌液37℃摇菌提质粒,测序。测序比对正确的质粒,用BamHI--XhoI进行双酶切,获得861bp、5372bp两个片段。
RBD-P2-6*HIS(GP101150-7)重组表达质粒抽提:接1%含RBD-P2-6*HIS(GP101150-7)重组表达质粒的大肠杆菌细胞(stbl3)于2mlLB培养基,37℃振荡培养过夜。处理细胞后,使用质粒提取试剂盒提取100ug质粒。测序比对正确的质粒,用BamHI--XhoI进行双酶切,获得861bp、5372bp两个片段。
实施例2细胞转染及RBD-P2-6*HIS蛋白纯化
在所有细胞操作过程中,温和地旋转混合宿主细胞,避免剧烈的混合/移液。继代培养和扩增CHO细胞(来自Thermo公司的EXPICHO)直到细胞密度达到4×10
6–6×10
6个/mL。第-1天:细胞扩增,扩增培养细胞到3×10
6–4×10
6个/mL,并允许细胞过夜生长。第0天:转染细胞,测定活细胞密度和存活率,细胞的密度应该达7×10
6–10×10
6个/mL,存活率到95–99%进行转染,新鲜的细胞表达培养基,预热至37℃,将细胞稀释至最终密度为6×10
6个/mL,50mm振幅培养箱90rpm进行37℃培养,8%CO
2。使用OPti-PRO SFM培养基制备转染试剂和RBD-P2-6*HIS(GP101150-7)重组表达质粒复合物(4℃),例:1ml CHO细胞准备40μl OPti-PRO SFM添加0.8ug RBD-P2-6*HIS(GP101150-7)重组表达质粒混匀放置5min;准备40μl OPti-PRO SFM添加3μl Expi Fectamine CHO试剂混匀放置5min,室温下1–5分钟,然后将溶液缓慢转移至摇瓶摇晃,在添加过程中轻轻摇动摇瓶。将细胞置于50mm振幅培养箱90rpm进行37℃培养,8%CO
2。转染后18-22小时,添加Enhancer和ExpiCHO Feed,执行标准实验方案。例:1ml细胞添加6μl Enhancer和0.24ml ExpiCHO Feed,将细胞置于50mm振幅培养箱90rpm进行37℃培养,8%CO
2。转染后8天收集,进行后续纯化。
蛋白纯化。培养基离心,上清添加Ni柱,振荡孵育2h,经过重力空柱管柱进行亲和层析纯化。平衡缓冲液:“PBS”,pH7.4,清洗10CV;清洗缓冲液:“PBS”,pH7.4 with20mM imidazole,清洗10CV;洗脱缓冲液:“PBS”,pH7.4 with 500mM imidazole,洗脱1CV,重复5次,得到RBD-P2-6*HIS纯化蛋白,如表2所示。
表2蛋白纯化后的浓度和体积
P101150-7 | Concentration(mg/ml) | Volume(ml) |
RBD-P2-6*HIS | 0.05 | 1 |
SDS-PAGE电泳与WB验证。A.1.0mm PAGE胶,上样量8μl,跑胶顺序:M.Molecular weight marker.Me.Culture medium.FT.Flow through.W.Wash.E.Eluted fractions。B.电泳:将RBD-P2-6*HIS纯化蛋白与上样缓冲液混合(RBD-P2-6*HIS纯化蛋白:5x上样缓冲液=4:1),煮沸5min,进行点样。首先100V恒压电泳,可以看到Marker逐渐变成一条细线。待Marker进入分离胶后,将电压调到300V继续电泳,直到蓝色的溴酚蓝条带到达胶的底部(约25min)。C.转膜,撬开玻璃板,小心取出凝胶。PVDF膜用甲醇活化30s后和滤纸剪成和胶块相同大小,在转膜液中浸透。从下往上依次为:滤纸-PVDF膜-凝胶-滤纸。注意各层之间尤其是胶和膜之间不能有气泡。将平皿中的转膜液全部倒进转膜盒中。20V恒压转移,50KDa蛋白需20min。分子量越大需时间越长。D.抗体孵育,转膜后取出PVDF膜,可以看到Marker转移到膜上。用5%脱脂奶(1g脱脂奶粉加100ml PBST)封闭1小时,用PBST漂洗2x5min。一抗(anti-His Mab)用5%脱脂奶粉(1g脱脂奶粉,100ml TBS)稀释,放在小盒中孵育,4℃脱色摇床中反应2h。取出PVDF膜,PBST漂洗4×10min。二抗(羊抗鼠)也用5%脱脂奶粉稀释,孵育1小时。PBST漂洗4x 10min。均使用摇床震动。E.显色,ECL显色:每一块PBST膜,用A液1mL和B液10μL混合后滴在膜上,5min后在化学发光成像系统中拍照记录,如图1所示。
实施例3构建NTD-P2-6*HIS(GP101150-8)重组表达质粒
信号肽添加:MFVFLVLLPLVS(SEQ ID NO:75),载体选择:pcdna3.1(+)。按照宿主CHO细胞进行密码子优化。NTD-P2-6*HIS的引物通过PCR的方法扩增得到片段PCR产物,通过多段重组的方法重组到目的载体pcdna3.1(+)(BamHI-XhoI酶切载体)中,得到NTD-P2-6*HIS(GP101150-8)重组表达质粒。反应体系如表3所示。
表3处理好的目的片段与载体连接反应体系
名称 | 体积 |
酶切后载体 | 5μl |
纯化的PCR产物 | 5μl |
无缝组装MIX | 10μl |
总体积 | 20μl |
转化的方法:将浓度在100ng/μl左右的NTD-P2-6*HIS(GP101150-8)重组表达质粒吸取1-3μl加入到100μl左右的感受态细胞里,轻轻晃动旋转以混匀,冰上放置3分钟。42℃水浴90s不要摇动;冰浴中放置3分钟左右;每管中加入500-800μl 37℃预温LB培养基,37℃摇床200rpm温和振荡40分钟。
NTD-P2-6*HIS(GP101150-8)重组表达质粒的验证:(1)制备含相应抗性的琼脂平板。(2)取100μl菌液,然后平铺在含有相应抗性的琼脂板,用无菌玻璃涂布器轻轻将细菌涂在平板表面,将平板置于37℃培养15分钟。(3)倒置平板于37℃培养12-16小时可出现菌落。(4)平板挑菌,37℃250转/分钟摇菌14小时,用菌液进行PCR鉴定,将阳性克隆菌液送测序。
克隆质粒的鉴定方法:PCR扩增NTD-P2-6*HIS片段,鉴定引物序列由公司内部引物部合成预期扩增的片段长度为1005bp,PCR反应采用20μL体系:引物0.5μL,模板菌液2μL,聚合酶缓冲液0.5μL,buffer3μl,ddH2O 14μL。循环参数:96℃预变性3min;95℃15S,58℃15S,72℃20s,23个循环,72℃终延伸1min。以菌液PCR方法筛选阳性克隆,获得的阳性菌液37℃摇菌提质粒,测序。测序比对正确的质粒,用BamHI--XhoI进行双酶切,获得1005bp、5372bp两个片段。
NTD-P2-6*HIS(GP101150-8)重组表达质粒抽提。接1%含NTD-P2-6*HIS(GP101150-8)重组表达质粒的大肠杆菌细胞(stbl3)于2mlLB培养基,37℃振荡培养过夜。处理细胞后,使用质粒提取试剂盒提取100ug质粒。测序比对正确的质粒,用BamHI--XhoI进行双酶切,获得1005bp、5372bp两个片段。
实施例4细胞转染及NTD-P2-6*HIS(GP101150-8)蛋白纯化
在所有细胞操作过程中,温和地旋转混合宿主细胞;避免剧烈的混合/移液。继代培养和扩增CHO细胞直到细胞密度达到4×10
6–6×10
6个/mL。第-1天:CHO细胞扩增,扩增培养细胞到3×10
6–4×10
6个/mL,并允许细胞过夜生长。第0天:转染细胞,测定活细胞密度和存活率,细胞的密度应该达7×10
6–10×10
6个/mL,存活率到95–99%进行转染,新鲜的细胞表达培养基,预热至37℃,将细胞稀释至最终密度为6×10
6个/mL,50mm振 幅培养箱90rpm进行37℃培养,8%CO
2。使用OPti-PRO SFM培养基制备转染试剂和NTD-P2-6*HIS(GP101150-8)重组表达质粒复合物(4℃),例:1ml细胞准备40μl OPti-PRO SFM添加0.8ug NTD-P2-6*HIS(GP101150-8)重组表达质粒混匀放置5min;准备40μl OPti-PRO SFM添加3μl Expi Fectamine CHO试剂混匀放置5min,室温下1–5分钟,然后将溶液缓慢转移至摇瓶摇晃,在添加过程中轻轻摇动摇瓶。将细胞置于50mm振幅培养箱90rpm进行37℃培养,8%CO
2。转染后18-22小时,添加Enhancer和ExpiCHO Feed,执行标准实验方案。例:1ml细胞添加6μl Enhancer和0.24ml ExpiCHO Feed,将细胞置于50mm振幅培养箱90rpm进行37℃培养,8%CO
2。转染后8天收集,进行后续纯化。
蛋白纯化,培养基离心,上清添加Ni柱,振荡孵育2h,经过重力空柱管柱进行亲和层析纯化。平衡缓冲液:“PBS”,pH7.4,清洗10CV;清洗缓冲液:“PBS”,pH7.4 with20mM imidazole,清洗10CV;洗脱缓冲液:“PBS”,pH7.4 with 500mM imidazole,洗脱1CV,重复5次,得到NTD-P2-6*HIS(GP101150-8)纯化蛋白,结果如表4所示。
表4 NTD-P2-6*HIS(GP101150-8)纯化蛋白的浓度和体积
P101150-8 | 浓度(mg/ml) | 体积(ml) |
NTD-P2-6*HIS | 0.03 | 1 |
SDS-PAGE电泳与WB验证。A.1.0mm PAGE胶,上样量8μl,跑胶顺序:M.Molecμlar weight marker.Me.Cμlture medium.FT.Flow through.W.Wash.E.Eluted fractions。B.电泳:将NTD-P2-6*HIS(GP101150-8)纯化蛋白与上样缓冲液混合(NTD-P2-6*HIS(GP101150-8)纯化蛋白:5x上样缓冲液=4:1),煮沸5min,进行点样。首先100V恒压电泳,可以看到Marker逐渐变成一条细线。待Marker进入分离胶后,将电压调到300V继续电泳,直到蓝色的溴酚蓝条带到达胶的底部(约25min)。C.转膜。撬开玻璃板,小心取出凝胶。PVDF膜用甲醇活化30s后和滤纸剪成和胶块相同大小,在转膜液中浸透。从下往上依次为:滤纸-PVDF膜-凝胶-滤纸。注意各层之间尤其是胶和膜之间不能有气泡。将平皿中的转膜液全部倒进转膜盒中。20V恒压转移,50KDa蛋白需20min。分子量越大需时间越长。D.抗体孵育。转膜后取出PVDF膜,可以看到Marker转移到膜上。用5%脱脂奶(1g脱脂奶粉加100ml PBST)封闭1小时,用PBST漂洗2x5min。一抗(anti-His Mab)用5%脱脂奶粉(1g脱脂奶粉,100ml TBS)稀释,放在小盒中孵育,4'C脱色摇床中反应2h。取出PVDF膜,PBST漂洗4×10min。二抗(羊抗鼠)也用5%脱脂奶粉稀释,孵育1小时。PBST漂洗4x10min。均使用摇床震动。E.显色。ECL显色:每一块PBST膜,用A液1mL和B液10μL混合后滴在膜上, 5min后在化学发光成像系统中拍照记录,如图2所示。
实施例5构建NTD-RBD-foldon-8*HIS重组表达质粒
信号肽添加:MFVFLVLLPLVS(SEQ ID NO:75),载体选择:pcdna3.1(+)。按照宿主CHO细胞进行密码子优化。NTD-RBD-foldon-8*HIS的引物通过PCR的方法扩增得到片段PCR产物,通过多段重组的方法重组到目的载体pcdna3.1(+)(BamHI-XhoI酶切载体)中,得到NTD-RBD-foldon-8*HIS重组表达质粒。反应体系如表5所示。
表5处理好的目的片段与载体连接反应体系
名称 | 体积 |
酶切后载体 | 5μl |
纯化的PCR产物 | 5μl |
无缝组装MIX | 10μl |
总体积 | 20μl |
转化的方法:将浓度在100ng/μl左右的NTD-RBD-foldon-8*HIS重组表达质粒吸取1-3μl加入到100μl左右的感受态细胞里,轻轻晃动旋转以混匀,冰上放置3分钟。42℃水浴90s不要摇动;冰浴中放置3分钟左右;每管中加入500-800μl 37℃预温LB培养基,37℃摇床200rpm温和振荡40分钟。
NTD-RBD-foldon-8*HIS重组表达质粒的验证:(1)制备含相应抗性的琼脂平板。(2)取100μl菌液,然后平铺在含有相应抗性的琼脂板,用无菌玻璃涂布器轻轻将细菌涂在平板表面,将平板置于37℃培养15分钟。(3)倒置平板于37℃培养12-16小时可出现菌落。(4)平板挑菌,37℃250转/分钟摇菌14小时,用菌液进行PCR鉴定,将阳性克隆菌液送测序。
克隆质粒的鉴定方法:PCR扩增NTD-RBD-foldon-8*HIS片段,鉴定引物序列由公司内部引物部合成预期扩增的片段长度为1761bp,PCR反应采用20μL体系:引物0.5μL,模板菌液2μL,聚合酶缓冲液0.5μL,buffer3μl,ddH
2O 14μL。循环参数:96℃预变性3min;95℃15S,58℃15S,72℃20s,23个循环,72℃终延伸1min。以菌液PCR方法筛选阳性克隆,获得的阳性菌液37℃摇菌提质粒,测序。测序比对正确的质粒,用BamHI--XhoI进行双酶切,获得1761bp、5372bp两个片段。
NTD-RBD-foldon-8*HIS重组表达质粒抽提。接1%含NTD-RBD-foldon-8*HIS重组表达质粒的大肠杆菌细胞(stbl3)于2mlLB培养基,37℃振荡培养过夜。处理细胞后,使用质粒提取试剂盒提取100ug质粒。测序比对正确的质粒,用BamHI--XhoI进行双酶切,获得 1761bp、5372bp两个片段。
实施例6细胞转染及蛋白纯化
在所有细胞操作过程中,温和地旋转混合宿主细胞;避免剧烈的混合/移液。继代培养和扩增CHO细胞直到细胞密度达到4×10
6–6×10
6个/mL。第-1天:细胞扩增,扩增培养细胞到3×10
6–4×10
6个/mL,并允许细胞过夜生长。第0天:转染细胞,测定活细胞密度和存活率,细胞的密度应该达7×10
6–10×10
6个/mL,存活率到95–99%进行转染,新鲜的细胞表达培养基,预热至37℃,将细胞稀释至最终密度为6×10
6个/mL,50mm振幅培养箱90rpm进行37℃培养,8%CO
2。
使用OPti-PRO SFM培养基制备转染试剂和NTD-RBD-foldon-8*HIS重组表达质粒复合物(4℃),例:1ml细胞准备40ul OPti-PRO SFM添加0.8ug NTD-RBD-foldon-8*HIS重组表达质粒混匀放置5min;准备40ul OPti-PRO SFM添加3ul Expi Fectamine CHO试剂混匀放置5min,室温下1–5分钟,然后将溶液缓慢转移至摇瓶摇晃,在添加过程中轻轻摇动摇瓶。将细胞置于50mm振幅培养箱90rpm进行37℃培养,8%CO
2。转染后18-22小时,添加Enhancer和ExpiCHO Feed,执行标准实验方案,例:1ml细胞添加6ul Enhancer和0.24ml ExpiCHO Feed,将细胞置于50mm振幅培养箱90rpm进行37℃培养,8%CO
2。转染后8天收集,进行后续纯化。
蛋白纯化:培养基离心,上清添加Ni柱,振荡孵育2h,经过重力空柱管柱进行亲和层析纯化。平衡缓冲液:“PBS”,pH7.4,清洗10CV;清洗缓冲液:“PBS”,pH7.4 with20mM imidazole,清洗10CV;洗脱缓冲液:“PBS”,pH7.4 with 500mM imidazole,洗脱1CV,重复5次,得到NTD-RBD-foldon-8*HIS纯化蛋白,结果如表6所示。
表6 NTD-RBD-foldon-8*HIS纯化蛋白的浓度和体积
P101150-5 | Concentration(mg/ml) | Volume(ml) |
NR-foldon-8*HIS | 0.05 | 0.5 |
SDS-PAGE电泳与WB验证。A.1.0mm PAGE胶,上样量8ul,跑胶顺序:M.Molecular weight marker.Me.Culture medium.FT.Flow through.W.Wash.E.Eluted fractions。B.电泳:将NTD-RBD-foldon-8*HIS纯化蛋白与上样缓冲液混合(NR-foldon-8*HIS纯化蛋白:5x上样缓冲液=4:1),煮沸5min,进行点样。首先100V恒压电泳,可以看到Marker逐渐变成一条细线。待Marker进入分离胶后,将电压调到300V继续电泳,直到蓝色的溴酚蓝条带到达胶的底部 (约25min)。C.转膜,撬开玻璃板,小心取出凝胶。PVDF膜用甲醇活化30s后和滤纸剪成和胶块相同大小,在转膜液中浸透。从下往上依次为:滤纸-PVDF膜-凝胶-滤纸。注意各层之间尤其是胶和膜之间不能有气泡。将平皿中的转膜液全部倒进转膜盒中。20V恒压转移,50KDa蛋白需20min。分子量越大需时间越长。D.抗体孵育转膜后取出PVDF膜,可以看到Marker转移到膜上。用5%脱脂奶(1g脱脂奶粉加100ml PBST)封闭1小时,用PBST漂洗2x5min。一抗(anti-His Mab)用5%脱脂奶粉(1g脱脂奶粉,100ml TBS)稀释,放在小盒中孵育,4'C脱色摇床中反应2h。取出PVDF膜,PBST漂洗4×10min。二抗(羊抗鼠)也用5%脱脂奶粉稀释,孵育1小时。PBST漂洗4x 10min.均使用摇床震动。E.显色,ECL显色:每一块PBST膜,用A液1mL和B液10μL混合后滴在膜上,5min后在化学发光成像系统中拍照记录,如图3所示。
实施例7构建载体和蛋白表达
参照上述实施例,构建如下需要表达的目的片段,并表达纯化蛋白,如表7所示。
表7不同的目的片段
基因名称 | 构建方式 | SEQ ID NO |
RBD-HBsAg-6*HIS | 全基因合成 | 45 |
NTD-HBsAg-6*HIS | 全基因合成 | 46 |
RBD-foldon-6*HIS | 全基因合成 | 47 |
NTD-foldon-6*HIS | 全基因合成 | 48 |
NTD-RBD-foldon-8*HIS | 全基因合成 | 49 |
S-foldon-8*HIS | 全基因合成 | 50 |
RBD-P2-6*HIS | 全基因合成 | 51 |
NTD-P2-6*HIS | 全基因合成 | 52 |
S1-P2-8*HIS | 全基因合成 | 53 |
RBD-P2-foldon-6*HIS | 全基因合成 | 54 |
NTD-P2-foldon-6*HIS | 全基因合成 | 55 |
S1-P2-foldon-6*HIS | 全基因合成 | 56 |
RBD-ferritin LC-6*HIS | 全基因合成 | 57 |
RBD-ferritin HC-FLAG | 全基因合成 | 58 |
RBD194-HBsAg | PCR克隆 | 59 |
NTD-ferritin HC核酸序列 | PCR克隆 | 60 |
NTD-ferritin LC核酸序列 | PCR克隆 | 61 |
NTD ferritin核酸序列 | 全基因合成 | 62 |
RBD ferritin核酸序列 | 全基因合成 | 63 |
实施例8疫苗组合物的制备
0.04mg/mL的RBD蛋白,0.04mg/mL的NTD蛋白;2%至15%(w/v)蔗糖;0.01%至0.05%(w/v)吐温80;和pH为5.0至7.0的5mM至25mM组氨酸缓冲液,分装到2ml管制瓶,每管0.5ml,置冻干机进行冷冻干燥。
实施例9疫苗组合物的制备
0.04mg/mL的RBD-P2蛋白,0.04mg/mL的NTD-P2蛋白;2%至15%(w/v)山梨糖醇;0.01%至0.05%(w/v)聚山梨酯20;和pH为5.5至7.0的5mM至20mM组氨酸缓冲液,分装到2ml管制瓶,每管0.5ml,置冻干机进行冷冻干燥。
实施例10疫苗组合物的制备
0.04mg/mL的RBD蛋白,0.04mg/mL的NTD-P2蛋白;2%至15%(w/v)蔗糖;0.01%至0.05%(w/v)吐温80;和pH为4.5至5.5的5mM至25m琥珀酸缓冲液,分装到2ml管制瓶,每管0.5ml,置冻干机进行冷冻干燥。
实施例11疫苗组合物的制备
0.04mg/mL的RBD-P2蛋白,0.04mg/mL的NTD蛋白;2%至15%(w/v)山梨糖醇;0.01%至0.05%(w/v)聚山梨酯20;和pH为5.5至7.0的5mM至20mM组氨酸缓冲液,分装到2ml管制瓶,每管0.5ml,置冻干机进行冷冻干燥。
实施例12疫苗组合物的制备
实施例8-11中任一项所述的免疫原性组合物,该组合物进一步包含氢氧化铝佐剂,浓度为1mg/mL。
实施例8-11中任一项所述的免疫原性组合物中加入CpG1018佐剂,浓度为6mg/mL,CpG佐剂(CpG由上海生工生物公司按CpG1018序列合成)。
实施例13疫苗组合物的制备
实施例8-11中任一项所述的免疫原性组合物,该组合物进一步包含佐剂,例如佐剂瓶为0.5ml,50μg的MPL,500μg氢氧化铝,150mM的NaCl,8mM的二水磷酸氢二钠,加注射用水至0.5ml。
实施例14疫苗组合物的制备
实施例8-11中任一项所述的免疫原性组合物,该组合物进一步包含佐剂,例如佐剂瓶为0.25ml,成份包括10.69mg的角鲨烯,11.86mg的α-生育酚,4.86mg的吐温80,3.53mg的NaCl,0.09mg的KCl,0.51mg的Na
2HPO
4,0.09mg的KH
2PO
4和注射用水。
实施例15疫苗组合物的制备
实施例8-11中任一项所述的免疫原性组合物,该组合物进一步包含佐剂,例如佐剂瓶为0.5ml,成份包括50μg的MPL,50μg的QS-21,1mg的DOPC二油酰磷脂酰胆碱,0.25mg的胆固醇,0.15mg的无水磷酸二钠,0.54mg的磷酸二氢钾,4.385mg的氯化钠,和注射用水。
实施例16疫苗组合物的制备
实施例8-11中任一项所述的免疫原性组合物,该组合物进一步包含佐剂,例如佐剂瓶为0.5ml,成份包括9.75mg角鲨烯、1.175mg司盘85、1.175mg吐温80、0.66mg二水合柠檬酸三钠和0.04mg一水合柠檬酸,和注射用水。
实施例17不同抗原与佐剂的小鼠免疫实验
抗原蛋白结合不同佐剂用于免疫小鼠,蛋白诱导的中和抗体水平通过ELISA测定确定。小鼠分组及免疫方案如表8所示。
表8小鼠分组表
其中的NTD蛋白序列为SEQ ID NO:20中1-291氨基酸,RBD的序列为SEQ ID NO:1中1-223氨基酸,Al(OH)
3佐剂、CpG佐剂、CpG+Al(OH)
3佐剂参照实施例12的浓度,AS01的组成参照实施例15,AS04的组成参照实施例13,小鼠免疫后28天后检测结果如表9所示。结果显示,RBD+NTD诱导产生中和抗体的水平更强。
表9小鼠免疫后28天后检测结果
实施例18不同抗原与佐剂的免疫原性评价实验
抗原蛋白与MF59佐剂共同用于免疫日本大耳朵白兔,蛋白诱导的中和抗体水平通过ELISA测定确定。日本大耳朵白兔分组及免疫方案如表10所示。
表10日本大耳白兔免疫分组表
NTD-P2的序列见SEQ ID NO:20,RBD-P2的序列参照SEQ ID NO:1,NTD-foldon的序列见SEQ ID NO:22,RBD-foldon的序列见SEQ ID NO:3,NTD-ferritin LC的序列见SEQ ID NO:23,RBD-ferritin的序列参照SEQ ID NO:5,NTD-HBsAg的序列参照SEQ ID NO:25,RBD-HBsAg的序列参照SEQ ID NO:6,佐剂MF59参照实施例16。
NTD-RBD-P2的序列参照SEQ ID NO:40序列,但C端无HIS标签,NTD-RBD-foldon的序列参照SEQ ID NO:41序列,但C端无HIS标签,NTD-RBD-P2-foldon的序列参照SEQ ID NO:42序列,但C端无HIS标签,NTD-RBD-ferritin的序列参照SEQ ID NO:43序列,NTD-RBD-HBsAg的序列参照SEQ ID NO:44序列。日本大耳朵白兔免疫28天后的检测结果如表11所示,说明融合蛋白具有免疫原性。
表11日本大耳白兔免疫28天后的检测结果
组别 | 抗原 | 佐剂 | 剂量 | 几何平均滴度(GMT) |
1 | NTD+RBD | MF59 | (10+10)μg | 71.84 |
2 | NTD-P2+RBD-P2 | MF59 | (10+10)μg | 203.2 |
3 | NTD-foldon+RBD-foldon | MF59 | (10+10)μg | 45.25 |
4 | NTD-ferritin+RBD-ferritin | MF59 | (10+10)μg | 128 |
5 | NTD-HBsAg+RBD-HBsAg | MF59 | (10+10)μg | 128 |
6 | NTD-RBD-P2 | MF59 | 20μg | 456.1 |
7 | NTD-RBD-foldon | MF59 | 20μg | 1024 |
8 | NTD-RBD-P2-foldon | MF59 | 20μg | 1024 |
9 | NTD-RBD-ferritin | MF59 | 20μg | 512 |
10 | NTD-RBD-HBsAg | MF59 | 20μg | 456.1 |
实施例19不同抗原与佐剂的免疫原性评价实验
抗原蛋白与不同佐剂共同用于免疫恒河猴,蛋白诱导的中和抗体水平通过ELISA测定确定。恒河猴分组及免疫方案如下,阳性对照为新冠的灭活苗。恒河猴免疫28天后的检测结果如表12所示。
表12恒河猴免疫结果
实施例20小鼠中重组新冠疫苗候选抗原诱导中和抗体产生的效果检测
以WIV04-1序列为基础,选择NTD和RBD作为候选抗原。检测单独NTD、单独RBD、NTD和RBD共免疫、NTD-RBD融合蛋白(以下简称NR),以及NTD-RBD-foldon融合蛋白(以下简称NR-foldon)的免疫原性,结果如表14所示。结果显示,NR-foldon抗原和BFA03佐剂联合免疫BALB/c小鼠后,能够显著有效诱导机体产生高滴度的中和抗体(基于SARS-CoV-2病毒株及VSV假病毒检测系统)和抗原特异性IgG抗体,其中,对武汉原型株以及D614G、Gamma突变株的中和效果显著优于其他抗原。
表13 BALB/c小鼠中重组新冠疫苗候选抗原实验设计概况
表14 BALB/c小鼠中重组新冠疫苗候选抗原的筛选结果
备注:1.基于SARS-CoV-2毒株的新冠病毒中和抗体检测,下同。
2.假病毒类型:原型株(WIV04-1)、主要流行毒株(D614G);VOC变异毒株:英国突变株(Alpha,B.1.1.7)、南非突变株(Beta,B.1.351)、巴西突变株(Gamma,P.1)、印度突变株(Delta,B.1.617.2);VOI变异毒株:秘鲁突变株(Lambda,C.37),下同。
实施例21不同佐剂的免疫效果检测
选择Al(OH)
3、BFA01、BFA02、BFA03、BFA04、CpG/Al(OH)
3与NR-foldon抗原配伍免疫BALB/c小鼠,检测血清中中和抗体及抗原特异性IgG抗体。研究结果表明,在候选佐剂中,BFA03佐剂的免疫效果最佳且显著优于传统铝佐剂(氢氧化铝佐剂),也优于以铝盐佐剂为基础的复合佐剂系统。
表15 BALB/c小鼠中重组新冠疫苗候选佐剂实验设计概况
备注:1.人用剂量(0.5ml)佐剂中主要成分的含量,BALB/c小鼠中的免疫剂量为1/5HD。
表16 BALB/c小鼠中重组新冠疫苗候选佐剂的实验结果
实施例22小鼠实验中不同剂量的免疫效果检测
将1/10及1/5人用剂量(即4μg和8μg)NR-foldon抗原,分别与1/5或1/10人用剂量BFA03佐剂联合免疫BALB/c小鼠。结果显示,在抗原剂量固定的前提下,1/10HD佐剂免疫产生中和抗体滴度低于1/5HD佐剂,各组不具有统计学差异,在一定程度上认为抗原和佐剂需要配比合适时可以诱导更强的免疫反应。
表17 BALB/c小鼠中重组新冠疫苗剂量考察结果
实施例23兔子实验中不同剂量的免疫效果检测
以1人用剂量BFA03佐剂(0.5ml)分别与20μg和40μg两个不同剂量的NR-foldon抗原联合免疫日本大耳白兔,结果如表18所示,40μg抗原免疫产生的抗体滴度较高。
表18不同剂量佐剂免疫日本大耳白兔结果
选择1HD(0.5ml)、1/2HD、1/4HD BFA03佐剂,分别与1HD NR-foldon抗原联合免疫日本大耳白兔,结果如表19所示,1HD的佐剂与1HD抗原免疫诱导机体产生的中和抗体及结合抗体水平最高,降低佐剂剂量则会降低免疫效果。
表19不同剂量佐剂免疫日本大耳白兔结果
实施例24抗原佐剂组合对新冠病毒不同变异株的中和作用检测
ReCOV疫苗(NR-foldon抗原/BFA03佐剂)免疫不同种属动物产生的中和抗体滴度GMT结果如下,由表可知,所确定的抗原佐剂组合对SARS-CoV-2病毒和不同的变异株均具有较好的中和作用。
表20不同种属动物产生的中和抗体滴度结果
使用Gamma突变株S蛋白NTD及RBD和Beta突变株S蛋白NTD及RBD的效果检测
实施例25构建NTD-RBD-foldon-6×His(Gamma突变株)和NTD-RBD-foldon-6×His(Beta突变株)重组表达质粒
如SEQ ID NO:85所示的NTD-RBD-foldon(Gamma突变株)添加信号肽MGWSCIILFLVATATGVHS(SEQ ID NO:91)和6×His;如SEQ ID NO:79所示的NTD-RBD-foldon(Beta突变株)添加信号肽MGWSCIILFLVATATGVHS(SEQ ID NO:91)和6×His,全长氨基酸序列由无锡药明生物技术股份有限公司进行密码子优化,并通过PCR进行全基因合成得到NTD-RBD-foldon-6×His(Gamma突变株)和NTD-RBD-foldon-6×His(Beta突变株)的DNA片段。经凝胶纯化后,将纯化后的DNA片段重组到目的载体pcDNA3.1(+)中,得到NTD-RBD-foldon-6×His(Gamma突变株)和NTD-RBD-foldon-6 ×His(Beta突变株)重组表达质粒。并通过Sanger测序验证,序列100%正确,测序结果见图1和图7。
转化宿主菌:(1)将浓度在100ng/μL的NTD-RBD-foldon-6×His(Gamma突变株)或NTD-RBD-foldon-6×His(Beta突变株)重组表达质粒吸取1~3μL加入到100μL的大肠杆菌E.coli Top10感受态细胞里,轻轻晃动旋转以混匀,冰上放置3分钟。(2)42℃水浴90s不要摇动。(3)冰浴中放置3分钟左右。(4)每管中加入500~800μL 37℃预温LB培养基,37℃摇床200rpm温和振荡40分钟。
重组表达质粒抽提并测序:(1)制备含100μg/mL氨苄青霉素的琼脂平板。(2)取100μL菌液培养后用无菌玻璃涂布器轻轻划LB琼脂平板(含100μg/mL氨苄青霉素),将平板置于37℃培养15分钟。(3)倒置平板于37℃培养12~16小时可出现菌落。(4)从平板上挑取一个单克隆接种至300mL LB培养基中扩大培养,采用NucleoBond Xtra Maxi EF试剂盒按照说明书进行质粒大量制备。并测序验证目的基因,测序结果与设计的基因组序列一致。
实施例26细胞转染及蛋白纯化
细胞转染:
在所有细胞操作过程中,温和地旋转混合宿主细胞,避免剧烈的混合/移液。继代培养和扩增CHO细胞(来自Thermo公司的ExpiCHO)直到细胞密度达到4×10
6~6×10
6个/mL。转染的第-1天:调整细胞密度到3×10
6~4×10
6个/mL,并允许细胞过夜生长。第0天:转染细胞,测定活细胞密度和存活率,细胞的密度应该达7×10
6~10×10
6个/mL,存活率达95~99%进行转染。新鲜的细胞表达培养基,预热至37℃,将细胞稀释至最终密度为6×106个/mL,在50mm振幅培养箱90rpm进行37℃培养,8%CO
2。使用OptiPRO
TM SFM培养基制备转染试剂和NTD-RBD-foldon-6×His(Gamma突变株/Beta突变株)重组表达质粒复合物(4℃),例:1mL CHO细胞准备40μL OptiPRO
TM SFM添加0.8μg NTD-RBD-foldon-6×His(Gamma突变株/Beta突变株)重组表达质粒,混匀放置5分钟;准备40μL OptiPRO
TM SFM添加3μL ExpiFectamineTM CHO试剂混匀放置5分钟,室温下1~5分钟,然后将溶液缓慢转移至摇瓶摇晃,在添加过程中轻轻摇动摇瓶。将细胞置于50mm振幅培养箱90rpm进行37℃培养,8%CO
2。转染后18-22小时,添加Enhancer和ExpiCHO Feed,执行标准实验方案。例:1mL细胞添加6μL Enhancer和0.24mL ExpiCHO Feed,将细胞置于50mm振幅培养箱90rpm进行37℃培养,8%CO
2。转染后6天收集,进行后续纯化。
蛋白纯化:
采用固定化金属离子亲和层析(IMAC)和尺寸排阻层析(SEC)相结合的方法进行纯化。采用装 载NiSO4的Hi Trap 5ml Chelating HP层析柱(美国GE公司)。鳌合层析介质预处理后装柱,分别用50mmol/L EDTA、0.2mol/L NaOH、超纯水洗柱。5倍以上的裂解缓冲液平衡后,细胞裂解上清直接上柱,以溶液A(20mmol/L PB,pH7.4,0.15mol/L NaCl)与溶液B(20mmol/L PB,pH7.4,0.15mol/L NaCl,0.5mol/L咪唑)为流动相进行梯度洗脱(溶液B的浓度从4%(体积)增大至100%(体积))。收集目标组分,继续进行SEC分离。采用HiLoad 26/60 Superdex-200 pre-grade凝胶柱(美国GE公司)。以溶液C(20mmol/L PB,pH7.4,0.15mol/L NaCl)为流动相洗脱1个柱体积,洗脱速率为2.5mL/min。SEC纯化后的层析图谱见图5和图8。
得到NTD-RBD-foldon-6×His(Gamma突变株/Beta突变株)纯化蛋白,蛋白纯化结果如表21所示,经纯化后的目的蛋白纯度可达93.6%(Gamma突变株)95%(Beta突变株)。
表21蛋白纯化结果
SDS-PAGE电泳和Western blot验证:
A.1.0mm PAGE胶,上样量8μL,跑胶顺序:M.Molecular weight marker.Me.Culture medium.FT.Flow through.W.Wash.E.Eluted fractions。B.电泳:将NTD-RBD-foldon(Gamma突变株/Beta突变株)纯化蛋白与上样缓冲液混合(NTD-RBD-foldon(Gamma突变株/Beta突变株)纯化蛋白:5×上样缓冲液=4:1),煮沸5分钟,进行点样。首先100V恒压电泳,可以看到Marker逐渐变成一条细线。待Marker进入分离胶后,将电压调到300V继续电泳,直到蓝色的溴酚蓝条带到达胶的底部(约25分钟)。C.转膜,撬开玻璃板,小心取出凝胶。PVDF膜用甲醇活化30s后和滤纸剪成和胶块相同大小,在转膜液中浸透。从下往上依次为:滤纸-PVDF膜-凝胶-滤纸。注意各层之间尤其是胶和膜之间不能有气泡。将平皿中的转膜液全部倒进转膜盒中。20V恒压转移,50KDa蛋白需20分钟。分子量越大需时间越长。D.抗体孵育,转膜后取出PVDF膜,可以看到Marker转移到膜上。用5%脱脂奶(1g脱脂奶粉加100mL PBST)封闭1小时,用PBST漂洗2×5min。一抗(anti-His Mab)用5%脱脂奶粉(1g脱脂奶粉, 100mL TBS)稀释,放在小盒中孵育,4℃脱色摇床中反应2h。取出PVDF膜,PBST漂洗4×10min。二抗(羊抗鼠)也用5%脱脂奶粉稀释,孵育1小时。PBST漂洗4×10min。均使用摇床震动。E.显色,ECL显色:每一块PBST膜,用A液1mL和B液10μL混合后滴在膜上,5分钟后在化学发光成像系统中拍照记录,如图6和图9所示。从SDS-PAGE电泳图和Western blot的结果图可知,经SEC分离后NTD-RBD-foldon(Beta突变株)溶液的电泳泳道上最大的条带是NTD-RBD-foldon(Beta突变株),NTD-RBD-foldon(Gamma突变株)溶液的电泳泳道上最大的条带是NTD-RBD-foldon(Gamma突变株),且其主要以三聚体的形式存在,Western blot结果可见对应的目的条带。
实施例27构建载体和蛋白表达
参照上述实施例,构建如表22所示需要表达的目的片段,并表达纯化蛋白。
表22不同的目的片段
实施例28免疫原性组合物的制备
50μg的NTD-RBD-foldon(Gamma突变株/Beta突变株)纯化蛋白,1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),分装到2mL管制瓶,每管0.5mL,置冻干机进行冷冻干燥。
50μg的NTD-RBD-foldon(Gamma突变株/Beta突变株)纯化蛋白,50μg的RBD-foldon(Beta突变株/Gamma突变株)纯化蛋白;1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),分装到2mL管制瓶,每管0.5mL,置冻干机进行冷冻干燥。
50μg的NTD-RBD-foldon(Gamma突变株/Beta突变株)纯化蛋白,50μg的NTD-foldon(Beta突变株/Gamma突变株)纯化蛋白;1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),分装到2mL管制瓶,每管0.5mL,置冻干机进行冷冻干燥。
50μg的NTD-RBD-foldon(Gamma突变株/Beta突变株)纯化蛋白,50μg的NTD-RBD-foldon(WIV04-1)纯化蛋白;1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),分装到2mL管制瓶,每管0.5mL,置冻干机进行冷冻干燥。
50μg的NTD-RBD-foldon(Gamma突变株/Beta突变株)纯化蛋白;135μg的HA蛋白(45μg的H1N1的HA蛋白,45μg的H3N2的HA蛋白,45μg的B/Washington/02/2019的HA蛋白);1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),分装到2mL管制瓶,每管0.5mL,置冻干机进行冷冻干燥。选用的HA蛋白如表23所示。
50μg的NTD-RBD-foldon(Gamma突变株/Beta突变株)纯化蛋白;180μg的HA蛋白(45μg的H1N1的HA蛋白,45μg的H3N2的HA蛋白,45μg的B/Washington/02/2019的HA蛋白,45μg的B/Phuket/3073/2013的HA蛋白);1%(w/v)蔗糖;2%(w/v)甘氨酸;0.02%(w/v)吐温80;和pH7.5的10mM的PB缓冲液(Na
2HPO
4,NaH
2PO
4),分装到2mL管制瓶,每管0.5mL,置冻干机进行冷冻干燥。选用的HA蛋白如表23所示。
表23联用的流感毒株
实施例29疫苗的制备
前述任一项实施例所述的免疫原性组合物,该组合物进一步包含氢氧化铝佐剂,浓度为1mg/mL。
前述任一项实施例所述的免疫原性组合物,进一步包含佐剂AS03,佐剂瓶为0.5mL,成分包括10.69mg的角鲨烯、11.86mg的α-生育酚、4.86mg的聚山梨酯80、3.53mg的氯化 钠、0.09mg的氯化钾、0.51mg的磷酸氢二钠、0.09mg的磷酸二氢钾和注射用水。
前述任一项实施例所述的免疫原性组合物,进一步包含佐剂MF59,佐剂瓶为0.5mL,成分包括4.5%的角鲨烯,0.5%Tween 80,0.5%Span85和注射用水。
实施例30疫苗的小鼠免疫实验(Beta突变株)
分别选用实施例28所述的免疫原性组合物结合AS03佐剂,按照2/25人用剂量进行小鼠免疫,以NTD-RBD-foldon(WIV04-1)蛋白制备的免疫原性组合物结合AS03佐剂作为对照,诱导的中和抗体水平通过基于VSV(水疱性口炎病毒)系统的新冠假病毒中和抗体检测方法测定。小鼠分组及免疫方案如表24所示。
表24小鼠免疫分组表
抗原(NTD-RBD-foldon) | 佐剂 | 抗原剂量 | 免疫程序 | 免疫途径 | 动物(只) |
Beta突变株 | AS03 | 4μg | 2剂次,间隔14d | 肌肉注射 | 10 |
WIV04-1 | AS03 | 4μg | 2剂次,间隔14d | 肌肉注射 | 10 |
WIV04-1+Beta突变株 | AS03 | (2+2)μg | 2剂次,间隔14d | 肌肉注射 | 10 |
AS03佐剂的配制参考实施例29,小鼠免疫第28天后,通过眼眶静脉采血的方式获得血清,利用基于VSV的假病毒检测系统检测中和抗体滴度,分别采用新冠病毒主要流行株(D614G)假病毒、新冠病毒Alpha突变株(B.1.1.7)假病毒、新冠病毒Beta突变株(501Y.V2)假病毒、新冠病毒Gamma突变株(501Y.V3)假病毒进行中和实验,考察疫苗对不同新冠病毒变异株的中和效果,检测结果如表25所示。结果显示,以NTD-RBD-foldon(WIV04-1)为抗原结合AS03佐剂诱导产生的中和抗体对主要流行株假病毒和Gamma突变株假病毒的中和效果较好(GMT远高于10000),而对于Alpha突变株假病毒和Beta突变株假病毒的中和效果较差(GMT远低于10000);以NTD-RBD-foldon(Beta突变株)为抗原结合AS03佐剂诱导产生的中和抗体对Beta突变株假病毒和Gamma突变株假病毒均有较好的中和效果(GMT远高于10000),更加令人意外的是,尤其是对于Gamma突变株假病毒的中和抗体GMT高达20944,但其对主要流行株假病毒和Alpha突变株假病毒的中和效果稍弱。可见,以NTD-RBD-foldon(Beta突变株)为免疫原的单价疫苗对4种新冠病毒突变株均有良好的免疫效果,其尤其可作为一种新的应对SARS-CoV-2突变株(尤其是南非和Gamma突变株)的COVID-19候选疫苗,更高效地诱导产生保护性中和抗体对抗新冠突变株的免疫逃逸。
而以NTD-RBD-foldon(Beta突变株)联合NTD-RBD-foldon(WIV04-1)为免疫原的二 价疫苗相比于NTD-RBD-foldon(Beta突变株)单价疫苗和NTD-RBD-foldon(WIV04-1)单价疫苗,诱导产生的中和抗体水平整体提升,既增强了NTD-RBD-foldon(WIV04-1)单价疫苗对Beta突变株假病毒的作用效果,又增强了NTD-RBD-foldon(Beta突变株)单价疫苗对主要流行株假病毒和英国流行株假病毒的作用效果。此二价疫苗具有作用范围更广,作用效果更强的特点,更适合作为一种应对COVID-19的候选疫苗发挥作用。
表25小鼠免疫28天后检测结果
实施例31疫苗的小鼠免疫实验(Gamma突变株)
选用实施例28所述的免疫原性组合物结合AS03佐剂,按照2/25人用剂量进行小鼠免疫,诱导的中和抗体水平通过基于VSV(水疱性口炎病毒)系统的新冠假病毒中和抗体检测方法测定。小鼠分组及免疫方案如表26所示。
表26小鼠免疫分组表
抗原 | 佐剂 | 抗原剂量 | 免疫程序 | 免疫途径 | 动物(只) |
NTD-RBD-foldon | AS03 | 4μg | 2剂次,间隔 | 肌肉注射 | 10 |
(Gamma突变株) | 14d |
AS03佐剂的配制参考实施例29,小鼠免疫第28天后,通过眼眶静脉采血的方式获得血清,利用基于VSV的假病毒检测系统检测中和抗体滴度,分别采用新冠病毒主要流行株(D614G)假病毒、新冠病毒Alpha突变株(B.1.1.7)假病毒、新冠病毒Beta突变株(501Y.V2)假病毒、新冠病毒Gamma突变株(501Y.V3)假病毒进行中和实验,考察疫苗对不同新冠病毒变异株的中和效果,检测结果如表27所示。结果显示,以NTD-RBD-foldon(Gamma突变株)为抗原结合AS03佐剂免疫小鼠诱导产生中和Beta突变株(501Y.V2)假病毒的抗体水平最高,诱导产生中和Gamma突变株(501Y.V3)假病毒的抗体水平其次,而诱导产生中和主要流行株(D614G)假病毒和Alpha突变株(B.1.1.7)假病毒的抗体水平较低。可见,以NTD-RBD-foldon(Gamma突变株)为免疫原的单价疫苗对4种新冠病毒突变株均有效,其可作为一种新的应对SARS-CoV-2突变株(尤其是南非和Gamma突变株)的COVID-19候选疫苗,更高效地诱导产生保护性中和抗体对抗新冠突变株的免疫逃逸。
表27小鼠免疫28天后检测结果
由以上结果可确定,若是按照上述方法制备NTD-RBD-foldon(Gamma突变株)联合NTD-RBD-foldon(Beta突变株)的二价疫苗,或NTD-RBD-foldon(Gamma突变株)联合NTD-RBD-foldon(WIV04-1)的二价疫苗,或NTD-RBD-foldon(Gamma突变株)联合NTD-RBD-foldon(Beta突变株)和NTD-RBD-foldon(WIV04-1)的三价疫苗,或NTD-RBD-foldon(Gamma突变株)联合NTD-RBD-foldon(Beta突变株)、NTD-RBD-foldon(WIV04-1)和流感血凝素HA的多价疫苗等多价联合疫苗可能会产生更高的中和抗体滴度。
Claims (45)
- 融合蛋白,其包含:1)SARS-CoV-2刺突蛋白(S蛋白)的受体结合结构域(RBD)或其功能活性片段;以及2)SARS-CoV-2刺突蛋白(S蛋白)N端结构域(NTD)或其功能活性片段。
- 根据权利要求1所述的融合蛋白,其中所述RBD和NTD源自SARS-CoV-2野生型或其突变体。
- 根据权利要求2所述的融合蛋白,其中所述SARS-CoV-2突变体选自下组中的任一种:Gamma突变体、Beta突变体、Alpha突变体和Delta突变体。
- 根据权利要求1-3中任一项所述的融合蛋白,其中所述RBD在选自下组的一个或多个氨基酸位点有突变:K417、L452、T478、E484和N501。
- 根据权利要求1-4中任一项所述的融合蛋白,其中所述RBD包含氨基酸突变K417T/N、L452R、T478K、E484K和N501Y的一种或多种。
- 根据权利要求1-5中任一项所述的融合蛋白,其中所述RBD包含K417T、E484K和N501Y氨基酸突变。
- 根据权利要求1-6中任一项所述的融合蛋白,其中所述RBD包含K417N、E484K和N501Y氨基酸突变。
- 根据权利要求1-7中任一项所述的融合蛋白,其中所述RBD包含SEQ ID NO:18、19、76、83、97-108中任一项所示的氨基酸序列。
- 根据权利要求1-8中任一项所述的融合蛋白,其中所述NTD包含在选自下组的一个或多个氨基酸位点的突变:L18、T19、T20、P26、D80、D138、R190、D215、L242-244和R246。
- 根据权利要求1-9中任一项所述的融合蛋白,其中所述NTD包含选自下组的一个或多个氨基酸突变:L18F、T19R、T20N、P26S、D80A、D138Y、R190S、D215G、L242-244del和R246I。
- 根据权利要求1-10中任一项所述的融合蛋白,其中所述NTD包含L18F、D80A、D215G、L242-244del和R246I突变。
- 根据权利要求1-11中任一项所述的融合蛋白,其中所述NTD包含L18F、T20N、P26S、D138Y和R246I氨基酸突变。
- 根据权利要求1-12中任一项所述的融合蛋白,其中所述NTD包含SEQ ID NO:37、38、77、84和109-111中任一项所示的氨基酸序列。
- 根据权利要求1-13中任一项所述的融合蛋白,其还包括一种或多种选自下组的多肽:P2 或其功能活性片段,foldon结构域或其功能活性片段,铁蛋白或其功能活性片段,以及乙肝表面抗原(HBsAg)或其功能活性片段。
- 根据权利要求1-14中任一项所述的融合蛋白,其中所述RBD或其功能活性片段与所述NTD或其功能活性片段直接或间接地相连。
- 根据权利要求1-15中任一项所述的融合蛋白,其中所述RBD或其功能活性片段与所述NTD或其功能活性片段在框内融合。
- 根据权利要求14-16中任一项所述的融合蛋白,其中所述多肽与所述RBD或其功能性片段和/或所述NTD或其功能活性片段直接或间接地相连。
- 根据权利要求14-17中任一项所述的融合蛋白,其中所述多肽与所述RBD或其功能活性片段和/或所述NTD或其功能活性片段在框内融合。
- 根据权利要求14-18中任一项所述的融合蛋白,其中所述多肽的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性片段的N端与所述NTD或其功能活性片段的C端直接或间接地相连。
- 根据权利要求14-19中任一项所述的融合蛋白,其中所述P2或其功能活性片段包含破伤风毒素的表位肽。
- 根据权利要求20所述的融合蛋白,其中所述破伤风毒素的表位肽包含SEQ ID NO:64-66中任一项所示的氨基酸序列。
- 根据权利要求14-21中任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段包含噬菌体T4纤维蛋白C末端的氨基酸残基。
- 根据权利要求14-22中任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段包含SEQ ID NO:67-69和78中任一项所示的氨基酸序列。
- 根据权利要求14-23中任一项所述的融合蛋白,其中所述foldon结构域或其功能活性片段的N端与所述P2或其功能活性片段的C端直接或间接地相连,所述P2或其功能活性片段的N端与所述RBD或其功能活性片段的C端直接或间接地相连,所述RBD或其功能活性的N端与所述NTD或其功能活性的C端直接或间接地相连。
- 根据权利要求14-24中任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含粉纹夜蛾铁蛋白、幽门螺杆菌铁蛋白或其功能活性片段。
- 根据权利要求25所述的融合蛋白,其中所述粉纹夜蛾铁蛋白或其功能活性片段包括粉纹夜蛾铁蛋白的重链或轻链。
- 根据权利要求26所述的融合蛋白,其中所述粉纹夜蛾铁蛋白的重链包含SEQ ID NO:70 所示的氨基酸序列,所述粉纹夜蛾铁蛋白的轻链包含SEQ ID NO:71所示的氨基酸序列。
- 根据权利要求14-27中任一项所述的融合蛋白,其中所述铁蛋白或其功能活性片段包含SEQ ID NO:70-72任一项所示的氨基酸序列。
- 根据权利要求14-28中任一项所述的融合蛋白,其中所述乙肝表面抗原或其功能活性片段包含SEQ ID NO:73所示的氨基酸序列。
- 根据权利要求15-29中任一项所述的融合蛋白,其中所述直接或间接相连包含通过连接子相连。
- 根据权利要求30所述的融合蛋白,其中所述连接子包含刚性接头,柔性接头或其他序列。
- 根据权利要求30-31中任一项所述的融合蛋白,其中所述连接子包含SEQ ID NO:89-90中任一项所示的氨基酸序列。
- 根据权利要求1-32中任一项所述的融合蛋白,其包含SEQ ID NO:39-44、79、85和96中任一项所示的氨基酸序列。
- 免疫原性组合物,其包含权利要求1-33中任一项所述的融合蛋白。
- 根据权利要求34所述的免疫原性组合物,其还包含至少一种人流感病毒血凝素蛋白HA。
- 根据权利要求35所述的免疫原性组合物,其中所述HA包含SEQ ID NO:92-95中任一项所述的氨基酸序列。
- 一种或多种分离的核酸分子,其编码权利要求1-33中任一项所述的融合蛋白。
- 根据权利要求37所述的核酸分子,其包含mRNA。
- 药物组合物,其包含权利要求1-33中任一项所述的融合蛋白,或权利要求34-36中任一项所述的免疫原性组合物,或权利要求37-38中任一项所述的核酸分子,及任选地药学上可接受的赋形剂。
- 权利要求1-33中任一项所述的融合蛋白,或权利要求34-36中任一项所述的免疫原性组合物,或权利要求37-38中任一项所述的核酸分子,在制备疫苗中的用途。
- 根据权利要求40所述的用途,其中所述疫苗用于预防和/或治疗COVID-19。
- 权利要求1-33中任一项所述的融合蛋白,或权利要求34-36中任一项所述的免疫原性组合物,或权利要求37-38中任一项所述的核酸分子,其用于治疗和/或预防COVID-19。
- 一种制备COVID-19亚单位疫苗的方法,其包括:1)提供权利要求1-33中任一项所述的融合蛋白,或权利要求34-36中任一项所述的免疫原性组合物,或权利要求37-38中任一项所述的核酸分子;以及2)使1)中所述的融合蛋白,免疫原性组合物或核酸分子与药学上可接受的佐剂混合。
- 一种检测SARS-CoV-2中和抗体的方法,其包括:1)向受试者施用权利要求43所述的COVID-19亚单位疫苗;以及2)检测1)中所述受试者在接受所述COVID-19亚单位疫苗后体内产生的中和抗体。
- 一种治疗和/或预防COVID-19的方法,其包括向受试者施用权利要求1-33任一项所述的融合蛋白,或权利要求34-36中任一项所述的免疫原性组合物,或权利要求37-38中任一项所述的核酸分子,或权利要求43所述的COVID-19亚单位疫苗。
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