Polypeptide carrier for presenting target polypeptide and uses thereof
10987418 · 2021-04-27
Assignee
Inventors
- Tianying Zhang (Xiamen, CN)
- Quan Yuan (Xiamen, CN)
- Xueran Guo (Xiamen, CN)
- Minxi Wei (Xiamen, CN)
- Xiaozhen Kang (Xiamen, CN)
- Jun Zhang (Xiamen, CN)
- Ningshao Xia (Xiamen, CN)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2730/10122
CHEMISTRY; METALLURGY
C12N2730/10134
CHEMISTRY; METALLURGY
A61K39/292
HUMAN NECESSITIES
C07K19/00
CHEMISTRY; METALLURGY
International classification
A61K39/39
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
C12N7/00
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
Abstract
The invention relates to a polypeptide carrier for presenting a target polypeptide, and use thereof. In particular, the invention relates to a nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide carrier, and being used for insertion of a nucleotide sequence encoding a target polypeptide. In addition, the invention further relates to a recombinant protein comprising the polypeptide carrier and a target polypeptide. Furthermore, the invention further relates to use of the nucleic acid molecule and the recombinant protein. In addition, the invention further relates to a vaccine or a pharmaceutical composition useful for preventing, alleviating or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B), comprising a recombinant protein comprising the polypeptide carrier of the invention and an epitope from HBV.
Claims
1. A nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide carrier, wherein the polypeptide carrier is selected from the following polypeptide carriers: (1) RBHBcAg-T carrier, which differs from roundleaf bat HBV core antigen protein (RBHBcAg protein) by difference comprising the following: (1a) one or more amino acid residues of the amino acid residues from positions 78-83 at N-terminus of RBHBcAg protein are deleted or substituted with a linker; (1b) one or more amino acid residues of the amino acid residues from positions 18-27, one or more amino acid residues of the amino acid residues from positions 50-69 and/or one or more amino acid residues of the amino acid residues from positions 120-140 at N-terminus of RBHBcAg protein are each independently substituted with a human T cell epitope; and (1c) optionally, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, or 35-40 amino acid residues are deleted at C-terminus of RBHBcAg protein; (2) TBHBcAg-T carrier, which differs from tent-making bat HBV core antigen protein (TBHBcAg protein) by difference comprising the following: (2a) one or more amino acid residues of the amino acid residues from positions 80-84 at N-terminus of TBHBcAg protein are deleted or substituted with a linker; (2b) one or more amino acid residues of the amino acid residues from positions 18-27, one or more amino acid residues of the amino acid residues from positions 54-73 and/or one or more amino acid residues of the amino acid residues from positions 124-144 at N-terminus of TBHBcAg protein are each independently substituted with a human T cell epitope; and (2c) optionally, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 30-35 amino acid residues are deleted at C-terminus of TBHBcAg protein; and (3) HBHBcAg-T carrier, which differs from horseshoe bat HBV core antigen protein (HBHBcAg protein) by difference comprising the following: (3a) one or more amino acid residues of the amino acid residues from positions 78-83 at N-terminus of HBHBcAg protein are deleted or substituted with a linker; (3b) one or more amino acid residues of the amino acid residues from positions 18-27, one or more amino acid residues of the amino acid residues from positions 50-69 and/or one or more amino acid residues of the amino acid residues from positions 120-140 at N-terminus of HBHBcAg protein are each independently substituted with a human T cell epitope; and (3c) optionally, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, or 35-40 amino acid residues are deleted at C-terminus of HBHBcAg protein.
2. The nucleic acid molecule according to claim 1, wherein (1) the polypeptide carrier is the RBHBcAg-T carrier; and optionally, the nucleic acid molecule is characterized by one or more of the following: (i) the RBHBcAg protein has an amino acid sequence as set forth in SEQ ID NO: 1; (ii) the amino acid residues from positions 78-83, positions 78-82, positions 78-81, or positions 78-80 at N-terminus of RBHBcAg protein are deleted or substituted with a linker; (iii) the linker is a flexible linker; (iv) the human T cell epitope is an MHC I or MHC II restricted human T cell epitope; (v) the human T cell epitope is selected from human T cell epitopes as set forth in SEQ ID NOs: 87-89; (vi) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid residues of the amino acid residues from positions 18-27 at N-terminus of RBHBcAg protein are substituted with an MHC I or MHC II restricted human T cell epitope; and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues of the amino acid residues from positions 50-69 at N-terminus of RBHBcAg protein are substituted with an MHC I or MHC II restricted human T cell epitope; and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acid residues of the amino acid residues from positions 120-140 at N-terminus of RBHBcAg protein are substituted with an MHC I or MHC II restricted human T cell epitope; (vii) the amino acid residues at positions 18-27, 50-69 and 120-140 at N-terminus of the RBHBcAg protein are substituted with human T cell epitopes set forth in SEQ ID NO: 87, 88 and 89, respectively; (viii) a restriction enzyme cleavage site is introduced at a position of nucleotides encoding the one or more amino acid residues within positions 78-83 at N-terminus of RBHBcAg protein that are deleted; (ix) a restriction enzyme cleavage site is introduced in the nucleotide sequence encoding the linker, and/or at either or both of the termini thereof; (x) the polypeptide carrier has an amino acid sequence as set forth in SEQ ID NO: 75 or 78; and (xi) the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 81 or 84; or (2) the polypeptide carrier is the TBHBcAg-T carrier; and optionally, the nucleic acid molecule is characterized by one or more of the following: (i) the TBHBcAg protein has an amino acid sequence as set forth in SEQ ID NO: 2; (ii) the amino acid residues from positions 80-84, positions 80-83, or positions 80-82, at N-terminus of TBHBcAg protein are deleted or substituted with a linker; (iii) the linker is a flexible linker; (iv) the human cell epitope is an MHC I or MHC II restricted human cell epitope; (v) the human T cell epitope is selected from human cell epitopes as set forth in SEQ ID NOs: 87-89; (vi) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid residues of the amino acid residues from positions 18-27 at N-terminus of TBHBcAg protein are substituted with an MHC 1 or MHC II restricted human cell epitope; and/or 1, 2; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues of the amino acid residues from positions 54-73 at N-terminus of TBHBcAg protein are substituted with an MHC I or MHC II restricted human T cell epitope; and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acid residues of the amino acid residues from positions 124-144 at N-terminus of TBHBcAg protein are substituted with an MHC 1 or MHC II restricted human T cell epitope; (vii) the amino acid residues at positions 18-27, 54-73 and 124-144 at N-terminus of the TBHBcAg protein are substituted with human T cell epitopes set forth in SEQ ID NO: 87, 88 and 89, respectively; (viii) a restriction enzyme cleavage site is introduced at a position of nucleotides encoding the one or more amino acid residues within positions 80-84 at N-terminus of TBHBcAg protein that are deleted; (ix) a restriction enzyme cleavage site is introduced in the nucleotide sequence encoding the linker, and/or at either or both of the termini thereof; (x) the polypeptide carrier has an amino acid sequence as set forth in SEQ ID NO: 76 or 79; and (xi) the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 82 or 85; or (3) the polypeptide carrier is the HBHBcAg-T carrier; and optionally, the nucleic acid molecule is characterized by one or more of the following: (i) the HBHBcAg protein has an amino acid sequence as set forth in SEQ NO: 3; (ii) the amino acid residues from positions 78-83, positions 78-82, positions 78-81, or positions 78-80 at N-terminus of HBHBcAg protein are deleted or substituted with a linker; (iii) the linker is a flexible linker; (iv) the human cell epitope is an MHC I or MHC II restricted human cell epitope; (v) the human T cell epitope is selected from human T cell epitopes as set forth in SEQ ID NOs: 87-89; (vi) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid residues of the amino acid residues from positions 18-27 at N-terminus of HBHBcAg protein are substituted with an MHC I or WIC II restricted human T cell epitope; and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues of the amino acid residues from positions 50-69 at N-terminus of HBHBcAg protein are substituted with an MHC I or MHC II restricted human T cell epitope; and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acid residues of the amino acid residues from positions 120-140 at N-terminus of HBHBcAg protein are substituted with an MHC I or MHC II restricted human T cell epitope; and (vii) the amino acid residues at positions 18-27, 50-69 and 120-140 at N-terminus of the HBHBcAg protein are substituted with human T cell epitopes set forth in SEQ ID NO: 87, 88 and 89, respectively; (viii) a restriction enzyme cleavage site is introduced at a position of nucleotides encoding the one or more amino acid residues within positions 78-83 at N-terminus of HBHBcAg protein that are deleted; (ix) a restriction enzyme cleavage site is introduced in the nucleotide sequence encoding the linker, and/or at either or both of the termini thereof; (x) the polypeptide carrier has an amino acid sequence as set forth in SEQ ID NO: 77 or 80; and (xi) the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 83 or 86.
3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule further comprises a nucleotide sequence encoding a target polypeptide, wherein the target polypeptide is heterologous relative to the polypeptide carrier, and the nucleotide sequence encoding the target polypeptide is inserted at a position of nucleotides encoding the one or more amino acid residues that are deleted, or is inserted in the nucleotide sequence encoding the linker or at either or both of the termini thereof.
4. The nucleic acid molecule according to claim 3, wherein the target polypeptide is an epitope peptide; optionally, the epitope peptide is characterized by one of the following: (i) the epitope peptide comprises: an epitope of HBsAg from human HBV, or an epitope of HIV GP120 protein; or an epitope of human PD-L1; (ii) the epitope peptide comprises: amino acids from positions 113-135 of HBsAg protein, or amino acids from positions 361-375 of HIV GP120 protein, or amino acids from positions 147-160 of human PD-L1 protein; and (iii) the epitope peptide has an amino acid sequence selected from SEQ ID NO: 20-22 and 60-62.
5. A vector, comprising the nucleic acid molecule according to claim 1.
6. A host cell, comprising the nucleic acid molecule according to claim 1 or a vector comprising the nucleic acid molecule.
7. A method for presenting a target polypeptide, comprising: (1) inserting a nucleotide sequence encoding the target polypeptide into the nucleic acid molecule according to claim 1, so as to obtain a nucleic acid molecule encoding a recombinant protein; wherein the nucleotide sequence encoding the target polypeptide is inserted at a position of nucleotides encoding the one or more amino acid residues that are deleted, or inserted in the nucleotide sequence encoding the linker or at either or both of the termini thereof; and (2) expressing the nucleic acid molecule encoding the recombinant protein in the step (1), to produce the recombinant protein.
8. The method according to claim 7, wherein the target polypeptide is an epitope peptide; optionally, the epitope peptide is characterized by one of the following: (i) the epitope peptide comprises: an epitope of HBsAg from human HBV, or an epitope of HIV GP120 protein, or an epitope of human PD-L1; (ii) the epitope peptide comprises: amino acids from positions 113-135 of HBsAg protein, or amino acids from positions 361-375 of HIV GP120 protein, or amino acids from positions 147-160 of human PD-L1 protein; and (iii) the epitope peptide has an amino acid sequence selected from SEQ ID NO: 20-22 and 60-62.
9. A recombinant protein, comprises a polypeptide carrier and a target polypeptide, wherein the target polypeptide is inserted at a position of the one or more amino acid residues that are deleted, or inserted in the linker or at either or both of the termini thereof; and the polypeptide carrier is selected from the following polypeptide carriers: (1) RBHBcAg-T carrier, which differs from roundleaf bat HBV core antigen protein (RBHBcAg protein) by difference comprising the following: (1a) one or more amino acid residues of the amino acid residues from positions 78-83 at N-terminus of RBHBcAg protein are deleted or substituted with a linker; (1b) one or more amino acid residues of the amino acid residues from positions 18-27, one or more amino acid residues of the amino acid residues from positions 50-69 and/or one or more amino acid residues of the amino acid residues from positions 120-140 at N-terminus of RBHBcAg protein are each independently substituted with a human T cell epitope; and (1c) optionally, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, or 35-40 amino acid residues are deleted at C-terminus of RBHBcAg protein; (2) TBHBcAg-T carrier, which differs from tent-making bat HBV core antigen protein (TBHBcAg protein) by difference comprising the following: (2a) one or more amino acid residues of the amino acid residues from positions 80-84 at N-terminus of TBHBcAg protein are deleted or substituted with a linker; (2b) one or more amino acid residues of the amino acid residues from positions 18-27, one or more amino acid residues of the amino acid residues from positions 54-73 and/or one or more amino acid residues of the amino acid residues from positions 124-144 at N-terminus of TBHBcAg protein are each independently substituted with a human T cell epitope; and (2c) optionally, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 30-35 amino acid residues are deleted at C-terminus of TBHBcAg protein; and (3) HBHBcAg-T carrier, which differs from horseshoe bat HBV core antigen protein (HBHBcAg protein) by difference comprising the following: (3a) one or more amino acid residues of the amino acid residues from positions 78-83 at N-terminus of HBHBcAg protein are deleted or substituted with a linker; (3b) one or more amino acid residues of the amino acid residues from positions 18-27, one or more amino acid residues of the amino acid residues from positions 50-69 and/or one or more amino acid residues of the amino acid residues from positions 120-140 at N-terminus of HBHBcAg protein are each independently substituted with a human T cell epitope; and (3c) optionally, 1-5, 5-10, 10-15, 15-20, 20-25, 5-30, 30-35, or 35-40 amino acid residues are deleted at C-terminus of HBHBcAg protein.
10. The recombinant protein according to claim 9, characterized by one or more of the following: (i) the polypeptide carrier has an amino acid sequence selected from SEQ. ID NO: 75-80: (ii) the target polypeptide is an epitope peptide; optionally, the epitope peptide comprises an epitope of HBsAg from human HBV, an epitope GP120 protein, or an epitope of human PD-L1; or comprises amino acids from positions 113-135 of HBsAg protein, amino acids from positions 361-375 of HIV GP120 protein, or amino acids from positions 147-160 of human PD-L1 protein; or comprises an amino acid sequence selected from SEQ ID NO: 20-22 and 60-62; and (iii) the recombinant protein comprises or consists of an amino acid sequence selected from SEQ ID NO: 90-96.
11. A virus-like particle, comprising the recombinant protein according to claim 9.
12. A pharmaceutical composition, comprising the recombinant protein according to claim 9 or a virus-like particle comprising the recombinant protein, and optionally, one or more pharmaceutically acceptable vehicles or excipients.
13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition is a vaccine; and/or, the pharmaceutically acceptable vehicles or excipient is an adjuvant.
14. A method for preventing or treating HBV infection or a disease associated with HBV infection, comprising, administering to a subject in need thereof the recombinant protein according to claim 9 or a virus-like particle comprising the recombinant protein, wherein the target polypeptide is an epitope peptide comprising an antigenic epitope from HBV.
15. The method according to claim 14, characterized by one or more of the following: (i) the disease associated with HBV infection is hepatitis B; (ii) the epitope peptide comprises an epitope of HBsAg from human HBV; (iii) the epitope peptide comprises amino acids from positions 113-135 of HBsAg protein of HBsAg from human HBV; (iv) the target polypeptide has an amino acid sequence selected from SEQ ID NO: 22 and 60-62; and (v) the recombinant protein comprises or consists of an amino acid sequence selected from SEQ ID NO: 90-96.
16. A polynucleotide, encoding the recombinant protein according to claim 9.
17. A vector, comprising the polynucleotide according to claim 16.
18. A host cell, comprising the polynucleotide according to claim 16 or a vector comprising the polynucleotide.
19. A method for preparing the recombinant protein according to claim 9, comprising culturing a host cell, which comprises a polynucleotide encoding the recombinant protein, under the condition suitable for expressing the recombinant protein, and, recovering the recombinant protein.
Description
DESCRIPTION OF THE DRAWINGS
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SEQUENCE INFORMATION
(18) Information on a part of sequences (SEQ ID NO: 1-44 and 75-96) involved in the invention is provided in the following Table 1.
(19) TABLE-US-00002 TABLE 1 Sequence information of SEQ ID NO: 1-44 and 75-96 SEQ ID NO Name Sequence information 1 RBHBcAg MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGPLIQDAIVQQVQASVGLRMRQLMWFHLSCLT FGQPTVIEFLVSFGTWIRTPQAYRPPNAPILSTLPEHTIVRRRGGSRATRSPRRRTP SPRRRRSQSPRRRRSQSPASSNC 2 TBHBcAg MENLERLDIYKEFGVSDVLVSPLPDDFFPTLQQLLESVNALYEDELTGPNHCSPH HTALRHLIMCGVELRDFIDWMHEQGLSPDADALLAGYLRSKYLKHITKAIWYH LSCLTFGKQTVHEYLVSFGTWIRTPAAYRPVNAPILTTLPETSVIRRRPASRRSTPS PRRRRSQSPRRRRSPSPRPASNC 3 HBHBcAg MDIDPYKEFGASSQLVSFLPADFFPALNDLVETTSVALYEEDLVGKEHCSPHHAAL RALLNCWEETVRLITWVRATVEGQPVQDAIIGYVQTTVGLRMRQQIWFHLSCLT FGQQTVIEFLVSFGTWMRTPAAYRPPNAPILSTLPEHTVIRRRGNPRAPRSPRRRT PSPRRRRSQSPRRRRSQSPAPSNC 4 RBHBcAg189 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIVQQ VQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRTPQAYRPPNAPILSTL PEHTIVRRRGGSRATRSPRRRTPSPRRRRSQSPRRRRSQSPASSNC 5 RBHBcAg149 MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIVQQ VQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRTPQAYRPPNAPILSTL PEHTIV 6 TBHBcAg188 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPK HTALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSEFGGGGSGGGGSDAD ALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGTWIRTPAAYRPVN APILTTLPETSVIRRRPASRRSTPSPRRRRSQSPRRRRSPSPRPASNC 7 TBHBcAg153 MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPH HTALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSEFGGGGSGGGGSDAD ALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGTWIRTPAAYRPVN APILTTLPETSVI 8 HBHBcAg189 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAAL RALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIIG YVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMRTPAAYRPPNAPILST LPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQSPRRRRSQSPAPSNC 9 HBHBcAg149 MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAAL RALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIIG YVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMRTPAAYRPPNAPILST 10 HBcAg183 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALR QAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSEFGGGGSGGGGSRELVVS YVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPQNAPILSTL PETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 11 HBcAg149 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALR QAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSEFGGGGSGGGGSRELVVS YVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPQNAPILSTL PETTVV 12 RBHBcAg189 ATGGACATTGATCCTTATAAAGAATTTGGAGCTTCATCTCAACTGATCTCTTTC TTGCCTGAGGACTTTTTCCCAAACCTTGCAGAATTGGTCGAGACCACCACAG CXCTCTATGAAGAAGAATTAGTAGGTAAGGAGCATTGCTCCCCTCACCATACT GCTTTACGATCCTTGCTAAATTGCTGGGGAGAGACTGTTAGATTAATAACTTG GGTCAGGAACTCTGTGGAGGGAGGTGGAGGTGGTTCTGGAGGTGGTGGTAC TGGATCCGAATTCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCCAAGATGCC ATTGTCCAGCAAGTTCAGGCCTCGGTGGGCCTGCGCATGAGACAGTTAATGT GGTTCCATCTCTCATGCCTAACATTTGGACAGCCCACTGTCATAGAATTTCTGG TCTCTTTTGGAACATGGATCAGAACCCCGCAAGCTTACAGACCCCCTAATGCA CCCATTCTCTCGACTGTTCCGGAGCATAGAATCGTTAGGAGAAGAGGAGGTTC ACGCGCTACTAGGTCCCCCCGAAGGCGCACTCCCTCTCCTCGCCGACGCAGA TCTCAATCGCCGCGTCGCCGCAGATCTCAGTCTCCAGCTTCCTCCAACTGCTA A 13 RBHBcAg149 ATGGACATTQATCCTTATAAAGAATTTGGAGCTTCATCTCAACTGATCTCTTTC TTGCCTGAGGACTTTTTCCCAAACCTTGCAGAATTGGTCGAGACCACCACAG CTCTCTATGAAGAAGAATTAGTAGGTAAGGAGCATTGCTCCCCTCACCATACT GCTTTACGATCCTTGCTAAATTGCTGGGGAGAGACTGTTAGATTAATAACTTG GGTCAGGAACTCTGTGGAGGGAGGTGGAGGTGGTTCTGGAGGTGGTGGTAC TGGATCCGAATTGGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCCAAGATGCC ATTGTCCAGCAAGTTCAGGCCTCGGTGGGCCTGCGCATGAGACAGTTAATGT GGTTCCATCTCTCATGCCTAACATTTGGACAGCCCACTGTCATAGAATTTCTGG TCTCTTTTGGAACATGGATCAGAACCCCGCAAGCTTACAGACCCCCTAATGCA CCCATTCTCTCGACTCTTCCGGAGCATACAATCGTT 14 TBHBcAg188 ATGGAAAACCTTGAAAGACTGACATCTATAAAGAATTTGGAGTCTCTGATGT CTTGGTGTCTTTCTTACCTGATGATTTCTTTCCAACTTTACAGCAACTTTTGGA ATCAGTGAATGCCCTATATQAGGATGAACTCACTGGGCCTAATCACTGTTCTC CCCATCATACTGCCTTAAGGCACTTGATTATGTGTGGGGTAGAATTAAGAGATT TTATTGATTGGATGCATGAACAGGGGGGTGGAGGTGGTTCTGGAGGTGGTGG TACTGGATCCGAATTCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCGATGCA GACGCTCTTTTGGCTGGTTACCTTCGATCCAAATATCTTAAACATATTACCAAG GGTATTTGGTATCATTTAAGGTGTTTGAGCTTTGGTAAGGAAAGAGTGCATGAA TACCTGGTATCCTTTGGCACCTGGATCAGAACCCCAGCTGCATATAGACCAGT GAATGCACCCATTCTCACCACTCTTCCGGAAACTTCAGTTATCAGAAGAAGG CCTGCCTCCAGAAGATCTACTCCCTCTCCTCGCAGACGCCGATCTCAATCACC GCGCCGCCGCCGCTCTCCATCTCCAAGACCAGCAAGCAATTGCTGA 15 TBHBcAg153 ATGGAAAACCTTGAAAGACTTGACATCTATAAAGAATTTGGAGTCTCTGATGT CTTGGTGTCTTTCTTACCTGATGATTTCTTTCCAACTTTACAGCAACTTTTGGA ATCAGTGAATGCGCTATATGAGGATGAAGTCACTGGGGCTAATGAGTGTTGTC CCCATCATACTGCCTTAAGGCACTTGATTATGTGTGGGGTAGAATTAAGAGATT TTATTGATTGGATGCATGAACAGGGGGGTGGAGGTGGTTCTGGAGGTGGTGG TACTGGATCCGAATTCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCGATGCA GACGCTCTTTTGGCTGGTTACCTTCGATCCAAATATCTTAAACATATTACCAAG GCTATTTGGTATCATTTAAGCTGTTTGACCTTTGGTAAGCAAACAGTGCATGAA TACCTGGTATCCTTTGGCAGCTGGATCAGAACCGCAGCTGCATATAGACCAGT GAATGCACCCATTCTCACCACTCTTCCGGAAACTTCAGTTATC 16 HBHBcAg189 ATGGACATTGATCCTTATAAAGAGTTCGGTGCTTCATCTCAACTTGTCTCCTTT TTGCCTGCTGACTTCTTTCCCGCCTTGAACGACCTGGTGGAAACTTCGGTGGC CTTATATGAGGAAGACCTTGTAGGTAAGGAGCATTGCTCCCCTCATCATGCAG CCTTAAGGGCCCTACTTAATTGCTGGGAGGAAACAGTCAGACTGATTACCTG GGTCCGTGCGACAGTAGAGGGAGGTGGAGGTGGTTCTGGAGGTGGTGGTAC TGGATCCGAATTCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCCAGGATGCC ATCATCGGTTATGTCCAGACTACGGTGGGCCTACGCATGAGACAACAGATCTG GTTCCATCTCTCATGCCTTACTTTTGGGCAGCAGACTGTGATAGAGTTCCTGG TCTCATTTGGGACATGGATGAGAACTCCAGCCGCCTATAGACCCCGCAATGCA CCCATTTTATCAACTCTTCCAGAGCACACAGTCATTAGGAGAAGAGGAAATGC GCGTGCTCCTAGGTCCCCCAGAAGGCGCACTCCCTCTCCTCGCCGACGCAGA TCTCAATCTCCGCGTCGCCGGAGATCTCAATCTCCAGCTCCCTCCAACTGCTA A 17 HBHBcAg149 ATGGACATTQATCCTTATAAAGAGTTCGGTGCTTCATCTCAACTTGTCTCCTTT TTGCCTGCTGACTTCTTTCCCGCCTTGAACGACCTGGTGGAAACTTCGGTGGC CTTATATGAGGAAGACCTTGTAGGTAAGGAGCATTGCTCCCCTCATCATGCAG CCTTAAGGGCCCTACTTAATTGCTGGGAGGAAACAGTCAGACTGATTACCTG GGTCCGTGCCACAGTAGAGGGAGGTGGAGGTGGTTCTGGAGGTGGTGGTAC TGGATCCGAATTCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCCAGGATGCC ATCATCGGTTATGTCCAGACTACGGTGGGCCTACGCATGAGACAACAGATCTG GTTCCATCTCTCATGCCTTACTTTTGGGCAGCAGACTGTGATAGAGTTCCTGG TCTCATTTGGGACATGGATGAGAACTCCAGCCGCCTATAGACCCCCCAATGCA CCCATTTTATCAACTCTTCCAGAGCACACAGTCATT 18 HBcAg183 ATGGACATTGATCCATATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTT TTGCCTTCCGACTTCTTTCCTTCTATCCGAGATCTCCTCGACACCGCCTCTGGT CTGTATGGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACGGC ACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTAGCCACCTGGG TGGGAAGTAATTTGGAAGATGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGG ATCCGAATTCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCAGGGAACTAGTA GTCAGCTATGTCAACGTTAATATGGGCCTAAAAATCAGACAACTATTGTGGTT TCACATTTCCTGTCTTACTTTTGGGAGAGAAAGTGTTGTTGAATATTTGGTGTG TTTTGGAGTGTGGATTCGCACTCCTCCTGCATATAGACCACAAAATGCCCCTA TCTTATCAACACTTCCGGAAACTACTGTTGTTCGTCGCCGAGGCCGTAGCCCG CGACGACGTACCCCGAGCCCGCGTCGACGTCGCAGCCAGAGCCCGCGCCGT CGTCGCAGCCAGAGCCGTGAAAGCCAGTGCTAA 19 HBcAg149 ATGGACATTGATCCATATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTT TTGCCTTCCGACTTCTTTCCTTCTATCCGAGATCTCCTCGACACCGCCTCTGCT CTGTATCGGGAGGGCTTAGAGTCTCCGGAACATTGTTCAGGTCAGCATACGGC ACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTAGCCACCTGGG TGGGAAGTAATTTGGAAGATGGTGGAGGTGGTTCTGGAGGTGGTGGTACTGG ATCCGAATTCGGTGGTGGAGGTTCAGGAGGAGGTGGTTCCAGGGAACTAGTA GTCAGCTATGTCAACGTTAATATGGGCCTAAAAATCAGACAACTATTGTGGTT TCACATTTGCTGTCTTACTTTTGGGAGAGAAAGTGTTCTTGAATATTTGGTGTC TTTTGGAGTGTGGATTCGCACTCCTCCTGCATATAGACCACAAAATGCCCCTA TCTTATCAACACTTCCGGAAACTACTGTTGTT 20 HIV-GP120- FKQSSGGDPEIVTHS aa 361-375 21 hPDL1- TSEHELTCQAEGYP aa147-160 22 HBsAg- SSTTSTGPCKTCTTPAQGTSMFP aa113-135 23 RBHBcAg189- MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ 20 SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGG GGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIR TPQAYRPPNAPTLSTLPEHTTVRRRGGSRATRSPRRRTPSPRRRRSQSPRRRRSQSP ASSNC 24 RBHBcAg149- MDIDPYKEFGASSQLLSFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ 20 SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGG GGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIR TPQAYRPPNAPILSTLPEHTIV 25 TBHBcAg188- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPH SEQ 20 HTALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSFKQSSGGDPEIVTHSEF GGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSF GTWIRTPAAYRPVNAPILTTLPETSVIRRRPASRRSTPSPRRRRSQSPRRRRSPSPRP ASNC 26 TBHBcAg153- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPH SEQ 20 HTALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSFKQSSGGDPEIVTHSEF GGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSF GTWIRTPAAYRPVNAPILTTLPETSVI 27 HBHBcAg189- MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAAL SEQ 20 RALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGG GGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMR TPAAYRPPNAPILSTLPEHTV1RRRGNPRAPRSPRRRTPSPRRRRSQSPRRRRSQSP APSNC 28 HBHBcAg149- MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAAL SEQ 20 RALLNCVVEETVRLITWVRATVEGGGGGSGGGGTGSFKQSSGGDPEIVTHSEFGG GGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMR TPAAYRPPNAPILSTLPEHTVI 29 RBHBcAg189- MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ 21 SLLNCWGETVRLJTWVRNSVEGGGGGSGGGGTGSTSEHELTCQAEGYPEFGGG GSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRT PQAYRPPNAPELSTLPEHTIVRRRGGSRATRSPRRRTPSPRRRRSQSPRRRRSQSPA SSNC 30 RBHBcAg149- MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ 21 SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSTSEHELTCQAEGYPEFGGG GSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGTWIRT PQAYRPPNAPJLSTLPEHTIV 31 TBHBcAg188- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPH SEQ 21 HTALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSTSEHELTCQAEGYPEF GGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSF GTWIRTPAAYRPVNAPILTTLPETSVIRRRPASRRSTPSPRRRRSQSPRRRRSPSPRP ASNC 32 TBHBcAg153- MENLERLDTYKEFGVSDVXVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPH SEQ 21 HTALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSTSEHELTCQAEGYPEF GGGGSGGGGSDADALLAGYLRSKYLKHITKArWYHLSCLTFGKQTVHEYLVSF GTWIRTPAAYRPVNAPILTTLPETSVI 33 HBHBcAg189- MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAAL SEQ 21 RALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSTSEHELTCQAEGYPEFGG GGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMR TPAAYRPPNAPfLSTLPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQSPRRRRSQSP APSNC 34 HBHBcAg149- MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAAL SEQ 21 RALLNCWEETVRUTWVRATVEGGGGGSGGGGTGSTSEHELTCQAEGYPEFGG GGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGTWMR TPAAYRPPNAPILSTLPEHTVI 35 HBHBcAg189- MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ 22 SLLNCWGETVRLITWVRNSVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTS MFPEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFL VSFGTWIRTPQAYRPPNAPILSTLPEHTIVRRRGGSRAIRSPRRRTPSPRRRRSQSP RRRRSQSPASSNC 36 RBHBcAg149- MDIDPYKEFGASSQLISFLPEDFFPNLAELVETTTALYEEELVGKEHCSPHHTALR SEQ 22 SLLNCVVGETVRLITWVRNSVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGTS MFPEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEFL VSFGTWIRTPQAYRPPNAPILSTLPEHTIV 37 TBHBcAg188- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPH SEQ 22 HTALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQ GTSMFPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQT VHEYLVSFGTWIRTPAAYRPVNAPILTTLPETSVIRRRPASRRSTPSPRRRRSQSPR RRRSPSPRPASNC 38 TBHBcAg153- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQLLESVNALYEDELTGPNHCSPH SEQ 22 HTALRHLIMCGVELRDFIDWMHEQGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQ GTSMFPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQT VHEYLVSFGTWIRTPAAYRPVNAPILTTLPETSVI 39 HBHBcAg189- MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAAL SEQ 22 RALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGT SMFPEFGGGGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFL VSFGTWMRTPAAYRPPNAPILSTLPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQS PRRRRSQSPAPSNC 40 HBHBcAg149- MDIDPYKEFGASSQLVSFLPADFFPALNDLVETSVALYEEDLVGKEHCSPHHAAL SEQ 22 RALLNCWEETVRLITWVRATVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGT SMFPEFGGGGSGGGGSQDAJTGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFL VSFGTWMRTPAAYRPPNAPILSTLPEHTVI 41 HBcAg183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALR SEQ 22 QAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGT SMFPEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYL VSFGVWIRTPPAYRPQNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRR SQSRESQC 42 HBcAg149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLIDTASALYREALESPEHCSPHHTALFI SEQ 22 QAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSSTTSTGPCKTCITPAQGT SMFPEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYL VSFGVWIRTPPAYRPQNAPILSTLPETTVV 43 Linker GGGGGSGGGGTGSEFGGGGSGGGGS 44 HBsAg MENIASGLLGPLLVLQAGFFLLTKILTIPQSLDSWWTSLNFLGGTPVCLGQNSQS QISSHSPTCCPPICPGYRWMCLRRFIIFLCILLLCLIFLLVLLDYQGMLPVCPLIPGS STTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASV RFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWFWGPSLYNILSPFMPLLPIFFCL WVYI 75 RBHBcAg189- MDJDPYKEFGASSQLISFLPSDFFPSVAELVETTTALYEEELVGKEHCSPHHTALR T3 QAILCWGELMTLATWVRNSVEGGGGCSGGGGTGSEFGGGGSGGCGSQDAIVQ QVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGVWIRTPPAYRPPNAPILST LPEHTVIRRRGGSRATRSPRRRTPSPRRRRSQSPRRRRSQSPASSNC 76 TBHBcAg188- MENLERDIYKEFGVSDFLPSDFFPSVFPTLQQLLESVNALYEDELTGPNHCSPH T3 HTALRQAILCWGELRDFIDWMHEQGGGGGSGGGGTGSEFGGGGSGGGGSDAD ALLAGYLRSKYLKHITKAIVVYHLSCLTFGKQTVHEYLVSFGVWIRTPPAYRPPN APILTTLPETSVIRRRPASRRSTPSPRRRRSQSPRRRRSPSPRPASNC 77 HBHBcAg189- MDIDPYKEFGASSQLVSFLPSDFFPSVNDLVETSVALYEEDLVGKEHCSPHHTAL T3 RQAILCWGELMTLATWVRATVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIIG YVQTTVGLRMRQQIWFHLSCLTFGQQTVIEFLVSFGVVYIRTPPAYRPPNAPILST LPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQSPRRRRSQSPAPSNC 78 RBHBcAg149- MDIDPYKEFGASSQLISFLPSDFFPSVAELVETTTALYEEELVGKEHCSPHHTALR T3 QAILCWGELMTLATWVRNSVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIVQ QVQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGVWIRTPPAYRPPNAPILST LPEHTVI 79 TBHBcAg153- MENLERLDIYKEFGVSDFLPSDFFPSVFPTLQQLLESVNALYEDELTCPNHCSPH T3 HTALRQAILCWGELRDFIDWMHEQGGGGGSGGGGTGSEFGGGGSGGGGSDAD ALLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHEYLVSFGVWIRTPPAYRPPN APILTTLPETSVI 80 HBHBcAg149- MDIDPYKEFGASSQLVSFLPSDFFPSVNDLVETSVALYEEDLVGKEHCSPHHTAL T3 RQAILCWGELMTLATWVRATVEGGGGGSGGGGTGSEFGGGGSGGGGSQDAIIG YVQTTVGLRMRQQIWFHLSCLTFGQQTV1EFLVSFGVWIRTPPAYRPPNAPILST LPEHTVI 81 RBHBcAg189- ATGGACATCGACCCGTACAAAGAATTCGGTGCTTCTTCTCAGCTGATCTCTTT T3 CCTGCCGTCTGACTTCTTCCCGTCTGTTGCTGAACTGGTTGAAACCACCACCG CTCTGTACGAAGAAGAACTGGTTGGTAAAGAACACTGCTCTCCGCACCACAC CGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGATGACCCTGGCTACC TGGGTTCGTAACTCTGTTGAAGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTA CCGGTTCTGAATTCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAGGACGC TATCGTTCAGCAGGTTCAGGCTTCTGTTGGTCTGCGTATGCGTGAGCTGATGT GGTTCCACCTGTCTTGCCTGACCTTCGGTCAGCCGACCGTTATCGAATTCCTG GTTTGTTTCGGTGTTTGGATCCGTAGGCCGCCGGCTTACCGTCCGCCGAAGGC TCCGATCCTGTCTACCCTGCCGGAACACACCGTTATCCGTCGTCGTGGTAAC CCGCGTGCTCCGCGTTCTCCGCGTCGTCGTACCCCGTCTCCGCGTCGTCGTCG TTCTCAGTCTCCGCGTCGTCGTCGTTCTCAGTCTCCGGCTCCGTCTAACTGCT AA 82 TBHBcAg188- ATGGAAAACCTGGAACGTCTGGACATCTACAAAGAATTCGGTGTTTCTGACT T3 TCCTGCCGTCTGACTTCTTCCCGTCTGTTTTCCCGACCCTGCAGCAGCTGCTG GAATCTGTTAACGCTCTGTACGAAGACGAACTGACCGGTCCGAACCACTGCT CTCCGCACCACACCGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGCG TGACTTCATCGACTGGATGCACGAACAGGGTGGTGGTGGTGGTTCTGGTGGT GGTGGTACCGGTTCTGAATTCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTG ACGCTGACGCTCTGCTGGCTGGTTACCTGCGTTCTAAATACCTGAAACACAT CACCAAAGCTATCTGGTACCACCTGTCTTGCCTGACCTTCGGTAAACAGACC GTTCACGAATACCTGGTTTCTTTCGGTGTTTGGATCCGTACCCCGCCGGCTTA CCGTCCGCCGAACGCTCCGATCCTGACCACCCTGCCGGAAAGCTCTGTTATC CGTCGTCGTCCGGCTTCTCGTCGTTCTACCCCGTCTCCGCGTCGTCGTCGTTC TCAGTCTCCGCGTCGTCGTCGTTCTCCGTCTCCGCGTCCGGCTTCTAACTGC 83 HBHBcAg189- ATGGACATCGACCCGTACAAAGAATTCGGTGCTTCTTCTCAGCTGGTTTCTTT T3 CCTGCCGTCTGACTTCTTCCCGTCTGTTAACGACCTGGTTGAAACCTCTGTTG CTCTGTACGAAGAAGACCTGGTTGGTAAAGAACACTGCTCTCCGCACCACAC CGCTCTGCCTCAGGCTATCCTGTGCTGGGGTGAACTGATGACCCTGGCTACC TGGGTTCGTGCTACCGTTGAAGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTA CCGGTTCTGAATTCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAGGACGC TATCATCGGTTACGTTCAGACCACCGTTGGTCTGCGTATGCGTCAGCAGATC TGGTTCCACCTGTCTTGCCTGACCTTCGGTCAGCAGACCGTTATCGAATTCCT GGTTTCriTCGGTGTTTGGATCCGTACCCCGCCGGCTTACCGTCCGGCGAACG CTCCGATCCTGTCTACCCTGCCGGAACACACCGTTATCCGTCGTCGTGGTAA CCCGCGTGCTCCGCGTTCTCCGCGTCGTCGTACCCCGTCTCCGCGTCGTCGTC GTTCTCAGTCTCCGCGTCGTCGTCGTTCTCAGTCTCCGGCTCCGTCTAACTGC 84 RBHBcAg149- ATGGACATCGACCCGTACAAAGAATTCGGTGCTTCTTCTCAGCTGATCTCTTT T3 CCTGCCGTCTGACTTCTTCCCGTCTGTTGCTGAACTGGTTGAAACCACCACCG CTCTGTACGAAGAAGAACTGGTTGGTAAAGAACACTGCTCTCCGCACCACAC CGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGATGACCCTGGCTACC TGGGTTCGTAACTCTGTTGAAGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTA CCGGTTCTGAATTCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAGGACGC TATCGTTCAGCAGGTTCAGGCTTCTGTTGGTCTGCGTATGCGTCAGCTGATGT GGTTCCACCTGTCTTGCCTGACCTTCGGTCAGCCGACCGTTATCGAATTCCTG GTTTCTTTCGGTGTTTGGATCCGTACCCCGCCGGCTTACCGTCCGCCGAACGC TCCGATCCTGTCTACCCTGCCGGAACACACCGTTATCTAA 85 TBHBcAg153- ATGGAAAACCTGGAACGTCTGGACATCTACAAAGAATTCGGTGTTTCTGACT T3 TCCTGCCGTCTGACTTCTTCCCGTCTGTTTTCCCGACCCTGCAGCAGCTGCTG GAATCTGTTAACGCTCTGTACGAAGACGAACTGACCGGTCCGAACCACTGCT CTCCGCACCACACCGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGCG TGACTTCATCGACTGGATGCACGAACAGGGTGGTGGTGGTGGTTCTGGTGGT GGTGGTAGCGGTTCTGAATTCGGTGGTGGTGGTTGTGGTGGTGGTGGTTCTG ACGCTGACGCTCTGCTGGCTGGTTACCTGCGTTCTAAATACCTGAAACACAT CACCAAAGCTATCTGGTACCACCTGTCTTGCCTGACCTTCGGTAAACAGACC GTTCACGAATACCTGGTTTCTTTCGGTGTTTGGATCCGTACCCCGCCGGCTTA CCGTCCGCCGAACGCTCCGATCCTGACCACCCTGCCGGAAACCTCTGTTATC 86 HBHBcAg149- ATGGACATCGACCCGTACAAAGAATTCGGTGCTTCTTCTCAGCTGGTTTCTTT T3 CCTGCCGTCTGACTTCTTCCCGTCTGTTAACGACCTGGTTGAAACCTCTGTTG CTCTGTACGAAGAAGACCTGGTTGGTAAAGAACACTGCTCTCCGCACCACAC CGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGATGACCCTGGCTACC TGGGTTCGTGCTACCGTTGAAGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTA CCGGTTCTGAATTCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAGGACGC TATCATCGGTTACGTTCAGACCACCGTTGGTCTGCGTATGCGTCAGCAGATC TGGTTCCACCTGTCTTGCCTGACCTTCGGTCAGCAGACCGTTATCGAATTCCT GGTTTCTTTCGGTGTTTGGATCCGTACCCCGCCGGCTTACCGTCCGCCGAACG CTCCGATCCTGTCTACCCTGCCGGAACACACCGTTATC 87 T cell epitope FLPSDFFPSV 88 T cell epitope PHHTALRQAILCWGELMTLA 89 T cell epitope VSFGVWIRTPPAYRPPNAPIL 90 RBHBcAg189- MDIDPYKEFGASSQLISFLPSDFFPSVAELVETTTALYEEELVGKEHCSPHHTALR T3-SEQ 22 QA1LCWGELMTLATWVRNSVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGT SMFPEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEF LVSFGVWIRTPPAYRPPNAPILSTLPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQ SPRRRRSQSPAPSNC 91 RBHBcAg149- MDIDPYKEFGASSQLISFLPSDFFPSVAELVETTTALYEEELVGKEHCSPHHTALR T3-SEQ 22 QAILCWGELMTLATWVRNSVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQGT SMFPEFGGGGSGGGGSQDAIVQQVQASVGLRMRQLMWFHLSCLTFGQPTVIEF LVSFGVWIRTPPAYRPPNAPILSTLPEHTVI 92 TBHBcAg188- MENLERLDIYKEFGVSDFLPSDFFPSVFPTLQQLLESVNALYEDELTGPNHCSPH T3-SEQ 22 HTALRQAILCWGELRDFIDWMHEQGGGGGSGGGGTGSSSTTSTGPCKTCTTPA QGTSMFPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQ TVHEYLVSFGVWIRTPPAYRPPNAPILTTLPETSVIRRRPASRRSTPSPRRRRSQSP RRRRSPSPRPASNC 93 TBHBcAg153- MENLERLDIYKEFGVSDFLPSDFFPSVFPTLQQLLESVNALYEDELTGPNHCSPH T3-SEQ 22 HTALRQAILCWGELRDFIDWMHEQGGGGGSGGGGTGSSSTTSTGPCKTCTTPA QGTSMFPEFGGGGSGGGGSDADALLAGYLRSKYLKHITKAIWYHLSCLTFGKQ TVHEYLVSFGVWIRTPPAYRPPNAPILTTLPETSVI 94 HBHBcAg189- MDIDPYKEFGASSQLVSFLPSDFFPSVNDLVETSVALYEEDLVGKEHCSPHHTAL T3-SEQ 22 RQAILCWGELMTLATWVRATVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQG TSMFPEFGGGGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEF LVSFGVWIRTPPAYRPPNAPILSTLPEHTVIRRRGNPRAPRSPRRRTPSPRRRRSQ SPRRRRSQSPAPSNC 95 HBHBcAg149- MDIDPYKEFGASSQLVSFLPSDFFPSVNDLVETSVALYEEDLVGKEHCSPHHTAL T3-SEQ 22 RQAILCWGELMTLATWVRATVEGGGGGSGGGGTGSSSTTSTGPCKTCTTPAQG TSMFPEFGGGGSGGGGSQDAIIGYVQTTVGLRMRQQIWFHLSCLTFGQQTVIEF LVSFGVWJRTPPAYRPPNAPILSTLPEHTVI 96 RBHBcAg149n- MDIDPYKEFGASSQLISFLPSDFFPSVAELVETTTALYEEELVGKEHCSPHHTALR T3-SEQ 22 QAILCWGELMTLATWVRNSVEGSSTTSTGPCKTCTTPAQGTSMFPQDAIVQQV QASVGLRMRQLMWFHLSCLTFGQPTVIEFLVSFGVWIRTPPAYRPPNAPILSTLP EHTVI
(20) Specific Modes for Carrying Out the Invention
(21) The invention is illustrated by reference to the following examples (which are intended to describe the invention rather than limiting the protection scope of the present invention).
(22) Unless indicated otherwise, the molecular biological experimental methods and immunological assays used in the present invention are carried out substantially in accordance with the methods as described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes are used under the conditions recommended by manufacturers of the products. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the protection scope of the present invention.
Example 1. Construction of Plasmids Encoding Polypeptide Carriers
(23) In the Example, plasmids encoding polypeptide carriers were constructed.
(24) 1.1 Preparation of Nucleotide Sequences Encoding Polypeptide Carriers
(25) Based on three bat-derived HBV core antigens (i.e., RBHBcAg protein, TBHBcAg protein, and HBHBcAg protein), the following polypeptide carriers were designed:
(26) RBHBcAg189 carrier, which differs from RBHBcAg protein (SEQ ID NO: 1) in that the amino acid residues from positions 78-81 of RBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid sequence of RBHBcAg189 carrier is set forth in SEQ ID NO: 4, and the nucleotide sequence of RBHBcAg189 carrier is set forth in SEQ ID NO: 12;
(27) TBHBcAg188 carrier, which differs from TBHBcAg protein (SEQ ID NO: 2) in that the amino acid residues from positions 80-83 of TBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid sequence of TBHBcAg188 carrier is set forth in SEQ ID NO: 6, the nucleotide sequence of TBHBcAg188 carrier is set forth in SEQ ID NO: 14;
(28) HBHBcAg189 carrier, which differs from HBHBcAg protein (SEQ ID NO: 3) in that the amino acid residues from positions 78-81 of HBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid sequence of HBHBcAg189 carrier is set forth in SEQ ID NO: 8, and the nucleotide sequence of HBHBcAg189 carrier is set forth in SEQ ID NO: 16.
(29) In addition, based on HBcAg protein of human HBV, HBcAg183 carrier was also designed, as a control. HBcAg183 carrier differs from HBcAg protein of human HBV in that the amino acid residues from positions 79-81 of HBcAg protein of human HBV are substituted with a linker set forth in SEQ ID NO: 43; the amino acid sequence of HBcAg183 carrier is set forth in SEQ ID NO: 10, and the nucleotide sequence of HBcAg183 carrier is set forth in SEQ ID NO: 18.
(30) With respect to the nucleotide sequences of said four carriers, their whole gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd.
(31) 1.2 Preparation of Plasmids Encoding Polypeptide Carriers
(32) By using the synthesized nucleotide sequences as templates, and using the primers in Table 2, the full-length genes and truncates (i.e., gene fragments truncated at C-terminus) of said 4 carriers were amplified by PCR, respectively. 8 PCR products were obtained, i.e., the gene encoding RBHBcAg189 carrier (SEQ ID NO: 12; the amino acid sequence encoded thereby is SEQ ID NO: 4), the gene encoding RBHBcAg149 carrier (SEQ ID NO: 13; the amino acid sequence encoded thereby is SEQ ID NO: 5), the gene encoding TBHBcAg188 carrier (SEQ ID NO: 14; the amino acid sequence encoded thereby is SEQ ID NO: 6), the gene encoding TBHBcAg153 (SEQ ID NO: 15; the amino acid sequence encoded thereby is SEQ ID NO: 7), the gene encoding HBHBcAg189 carrier (SEQ ID NO: 16; the amino acid sequence encoded thereby is SEQ ID NO: 8), the gene encoding HBHBcAg149 carrier (SEQ ID NO: 17; the amino acid sequence encoded thereby is SEQ ID NO: 9), the gene encoding HBcAg183 carrier (SEQ ID NO: 18; the amino acid sequence encoded thereby is SEQ ID NO: 10), and the gene encoding HBcAg149 carrier (SEQ ID NO: 19; the amino acid sequence encoded thereby is SEQ ID NO: 11).
(33) pTO-T7 vector (Luo Wenxin, Zhang Jun, Yang Haijie, et al., Construction and Application of an Escherichia coli High Effective Expression Vector with an Enhancer [J], Chinese Journal of Biotechnology, 2000, 16(5): 578-581) was subjected to double enzyme digestion by NdeI and HindIII, to obtain a linear vector. By Gibson assembly cloning method (New England Biolabs (UK) Ltd), 8 PCR products obtained were ligated to the linear vector, and transformed into DH5a competent bacteria. The transformed bacteria were spread on a plate and cultured, monoclonal colonies were then selected, and the plasmids were extracted and sequenced. It was confirmed by sequencing that 8 plasmids comprising the nucleotide sequences encoding the polypeptide carriers were obtained.
(34) The primers involved in the PCR are shown in Table 2.
(35) TABLE-US-00003 TABLE 2 Primer sequences SEQ ID NO: Primer name Sequence 45 RBHBcAg149/189F ACTTAAGAAGGAGATATACATATGATGGA CATTGATCCTTATAAAG 46 RBHBcAg149R GTGGTGCTCGAGGCGGCCGCAAGCTTTTA AACGATTGTATGCTCCGGAAGAGTCGA 47 RBHBcAg189R GTGGTGCTCGAGGCGGCCGCAAGCTTTTA GCAGTTGGAGGAAGCTGGAGACTGAGATC TGCGGCGAC 48 TBHBcAg153/188F ACTTTAAGAAGGAGATATACATATGATGG AAAACCTTGAAAGACTTG 49 TBHBcAg153R GTGGTGCTCGAGGCGGCCGCAAGCTTTTA GATAACTGAAGTTTCCGGAAGAGTG 50 TBHBcAg188R GTGGTGCTCGAGGCGGCCGCAAGCTTTTA GCAATTGCTTGCTGGTCTTG 51 HBHBcAg149/189F ACTTTAAGAAGGAGATATACATATGATGG ACATTGATCCTTATAAAG 52 HBHBcAg149R GTGGTGCTCGAGGCGGCCGCAAGCTTTTA AATGACTGTGTGCTCTGGAAGAGTTGA 53 HBHBcAg189R GTGGTGCTCGAGGCGGCCGCAAGCTTTTA GCAGTTGGAGGGAGCTGGAGATTGAGATC TCCGGCGAC
Example 2. Preparation of Recombinant Proteins
(36) In the Example, a nucleotide sequence encoding a target polypeptide was inserted into the plasmid constructed in Example 1, and a recombinant protein comprising the target polypeptide and the polypeptide carrier was obtained. The scheme of cloning solutions, in which recombinant proteins are constructed by inserting a target polypeptide (a target antigen peptide fragment) into RBHBcAg carrier, TBHBcAg carrier and HBHBcAg carrier of the invention, is shown in
(37) 2.1 Construction of Expression Plasmids of Recombinant Proteins Comprising a Target Polypeptide and a Polypeptide Carrier
(38) In the Example, 3 target polypeptides were used to verify the versatility of the polypeptide carrier of the invention for presenting peptide fragments. Said 3 target polypeptides were: polypeptide HIV-GP120-aa361-375 (i.e., the amino acids from positions 361-375 of HIV GP120 protein, its amino acid sequence is set forth in SEQ ID NO: 20); polypeptide hPDL1-aa147-160 (i.e., the amino acids from positions 147-160 of human PD-L1 protein, its amino acid sequence is set forth in SEQ ID NO: 21); and polypeptide HBsAg-aa113-135 (i.e., the amino acids from positions 113-135 of hepatitis B surface antigen (HBsAg) from human HBV, its amino acid sequence is set forth in SEQ ID NO: 22).
(39) The sense and antisense sequences coding said 3 target polypeptides (as shown in Table 3) were synthesized directly, and annealed, so as to obtain the gene fragments having cohesive end and encoding the target polypeptides.
(40) TABLE-US-00004 TABLE 3 Sense and antisense sequences coding 3 target polypeptides SEQ ID NO: Primer name Sequence 54 hPDL1-aa147-160F GATCCACCTCTGAACATGAACTGA CATGTCAGGCTGAGGGCTACCCCG 55 hPDL1-aa147-160R AATTCGGGGTAGCCCTCAGCCTGA CATGTCAGTTCATGTTCAGAGGTG 56 HIV-GP120-aa361-375F GATCCTTCAAACAGTCTTCTGGTGG TGACCCGGAAATCGTTACCCACTCTG 57 HIV-GP120-aa361-375R AATTCAGAGTGGGTAACGATTTCCG GGTCACCACCAGAAGACTGTTTGAAG 58 HBsAg-aa113-135F GATCCTCATCAACAACCAGCACCGG ACCATGCAAAACCTGCACAACTCCT GCTCAAGGAACCTCTATGTTTCCCG 59 HBsAg-aa113-135R AATTCGGGAAACATAGAGGTTCCTT GAGCAGGAGTTGTGCAGGTTTTGCAT GGTCCGGTGCTGGTTGTTGATGAG
(41) The 6 plasmids (RBHBcAg189, RBHBcAg149, TBHBcAg188, TBHBcAg153, HBHBcAg189 and HBHBcAg149) obtained in Example 1 were subjected to double enzyme digestion by BamHI and EcoRI, to obtain 6 linear vectors. Then, the 3 gene fragments having cohesive end and encoding the target polypeptides, as prepared above, were ligated to the linear vectors, to obtain the expression plasmids encoding recombinant proteins (18 in total: RBHBcAg189-SEQ20, RBHBcAg149-SEQ20, TBHBcAg188-SEQ20, TBHBcAg153-SEQ20, HBHBcAg189-SEQ20, HBHBcAg149-SEQ20, RBHBcAg189-SEQ21, RBHBcAg149-SEQ21, TBHBcAg188-SEQ21, TBHBcAg153-SEQ21, HBHBcAg189-SEQ21, HBHBcAg149-SEQ21, RBHBcAg189-SEQ22, RBHBcAg149-SEQ22, TBHBcAg188-SEQ22, TBHBcAg153-SEQ22, HBHBcAg189-SEQ22, and HBHBcAg149-SEQ22).
(42) 2.2 Expression, Purification and Assembly of Recombinant Proteins
(43) The 18 expression plasmids constructed in the previous step, were used to express and purify the recombinant proteins encoded by the expression plasmids via the same method. RBHBcAg149-SEQX (SEQX represents SEQ20, SEQ21 or SEQ22) was used as an example to describe the expression and purification of the recombinant proteins.
(44) (2.2.1) Preparation of bacterial strains for expressing recombinant proteins: the expression plasmid RBHBcAg149-SEQX obtained in 2.1 was transformed into E. coli strain ER2566, so as to obtain the expression bacterial strain.
(45) (2.2.2) Expression of the recombinant protein RBHBcAg149-SEQX: the expression bacterial strain was seeded in a 500 mL triangular flask, and was cultured at 37° C. on a shaking table until OD was about 1.0; later, isopropyl-beta-D-thiogalactoside (IPTG) was added at a final concentration of 0.5 mM, and the expression was further performed at 25° C. for 6 h.
(46) (2.2.3) Purification of the recombinant protein RBHBcAg149-SEQX:
(47) (2.2.3.1) Ultrasonic disruption of bacteria: the bacteria in 2.2.2 were harvested by centrifugation, and were subjected to ultrasonic disruption. Sonication buffer: 20 mM phosphate buffer (PH6.0)+300 mM NaCl.
(48) (2.2.3.2) Primary purification of the recombinant protein: the mixture obtained after ultrasonic disruption was incubated in a 65° C. water bath for 30 min, and the supernatant was then collected by centrifugation; saturated ammonium sulfate was added to the supernatant at a volume ratio of 1:1, and the precipitate was collected by centrifugation a suitable volume of buffer (20 mM phosphate buffer (pH=7.4)+150 mM NaCl) was added to resuspend the precipitate, so as to obtain the primarily purified recombinant protein RBHBcAg149-SEQX.
(49) (2.2.3.3) Purification of the recombinant protein by chromatography: in accordance with the instructions of manufacturer, the protein obtained in 2.2.3.2 was further purified by Sepharose 4FF(GE) molecular sieve column chromatography, so as to obtain the purified recombinant protein. The purified target protein was detected by SDS-PAGE, and the VLP formed by the recombinant protein was observed by Transmission Electron Microscope (TEM).
(50)
Example 3. Evaluation on Immunogenicity of Virus-Like Particles
(51) In the Example, the inventors verified the immunogenicity of the virus-like particles formed by the recombinant proteins prepared in Example 2. All such virus-like particles can induce generation of antibodies that specifically bind to target antigens in organisms.
(52) 3.1 Immunization of Mice
(53) BALB/C mice were immunized with the 18 virus-like particles prepared in Example 2, respectively. The immunization process was as followed: the immunoadjuvant used was aluminum hydroxide adjuvant; the immunizing dose was 3 ug/dose; the immunization was performed by intramuscular injection at lateral thigh of hindlimb; the immune procedure was primary immunization+booster immunization 2 weeks later (i.e. two times in total).
(54) 3.2 Detection of Titer of Antibodies that Specifically Bind to Target Antigens in Sera
(55) 3.2.1 Preparation of Reaction Plates
(56) The antigens for coating reaction plates were the target antigens corresponding to said three target polypeptides, i.e., HIV-1 gp120 protein (purchased from Sino Biological Inc., Catalog No. 11233-V08H), human PD-L1 protein (purchased from Sino Biological Inc.), and human hepatitis B virus surface antigen recombinantly expressed in CHO cells (HBsAg, purchased from Beijing Wantai Biological Pharmacy).
(57) 3 recombinant proteins were diluted with pH9.6 50 mM CB buffer (NaHCO.sub.3/Na.sub.2CO.sub.3 buffer, at a final concentration of 50 mM, pH=9.6), respectively, at a final concentration of 2 μg/mL, to obtain the coating solutions. To each well of a 96-well ELISA plate, 100 μL coating solution was added, and the wells were coated at 2-8° C. for 16-24 h, and then further coated at 37° C. for 2 h. After that, PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) was used to wash wells once; and 200 μL blocking solution (20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer solution containing 20% bovine calf serum and 1%/0 casein, pH=7.4) was then added to each well, and the wells were blocked at 37° C. for 2 h. The blocking solution was discarded. After that, the ELISA plate was dried, and packaged into an aluminum foil bag, which was stored at 2-8° C. for further use.
(58) 3.2.2 ELISA Detection of Anti-HBsAg Antibody Titer in Serum
(59) Collection of serum samples: blood was collected from the eye orbit of mice at Week 0, 2, and 4, the serum was separated and cryopreserved at −20° C., until detection.
(60) Sample dilution: a mouse serum was diluted with PBS solution containing 20% newborn bovine serum at 7 dilution gradients, i.e. 1:100, 1:500, 1:2500, 1:12500, 1:62500, 1:312500, and 1:1562500.
(61) ELISA detection: to each well of the coated ELISA plate, 100 μL diluted serum sample was added, and incubated at 37° C. for 30 min. The ELISA plate was then washed with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for five times. After washing, to each well of the ELISA plate, 100 μL GAM-HRP reaction solution was added, and incubated at 37° C. for 30 min. The ELISA plate was then washed with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for five times. After washing, to each well of the ELISA plate, 50 μL TMB color developing agent (provided by Beijing Wantai Biological Pharmacy) was added, and incubated at 37° C. for 15 min. After the incubation, to each well of the ELISA plate, 50 μL stop solution (provided by Beijing Wantai Biological Pharmacy) was added, and the OD450/630 value for each well was read by an ELISA instrument.
(62) Calculation of antibody titer: samples, the read values of which were within 0.2-2.0, were analyzed; a regression curve was plotted with the dilution fold and the read value, and the dilution fold of the sample, at which the read value was 2-fold of the background value, was calculated; and the dilution fold of the sample was used as the titer of the specific antibody in serum.
(63)
Example 4. Evaluation on Anti-HBV Therapeutic Effects of Virus-Like Particles Presenting HBsAg Epitope (SEQ ID NO: 22)
(64) In the Example, the inventors evaluated the anti-HBV therapeutic effects of the virus-like particles presenting the same epitope peptide (SEQ ID NO: 22), as constructed based on different polypeptide carriers.
(65) 4.1 Immunization of Mice
(66) According to the methods described in Example 1-2, 2 recombinant proteins (i.e., HBcAg183-SEQ22, its amino acid sequence is set forth in SEQ ID NO: 41; and HBcAg149-SEQ22, its amino acid sequence is set forth in SEQ ID NO: 42), presenting HBsAg epitope (SEQ ID NO: 22) and constructed based on HBcAg of human HBV, were prepared, and the virus-like particles formed by the 2 recombinant proteins were prepared.
(67) Later, 5 virus-like particles presenting HBsAg epitope (SEQ ID NO: 22) prepared in Example 2, and 2 virus-like particles prepared in the Example were evaluated for the anti-HBV therapeutic effects in a HBV transgenic mouse model.
(68) The immunization method was as followed: the immunoadjuvant used was aluminum hydroxide adjuvant; and the immunizing dose was 12 μg/dose; the immunization was performed by intramuscular injection at lateral thigh of hindlimb: the immune procedure was immunization at Week 0, 2, 3, 4, 5, and 6, (i.e. six times in total).
(69) 4.2 Detection of Antibody Titer and Virological Index in Serum
(70) According to the method as described in Example 3.2, the Anti-HBsAg antibody titer in serum was determined, and the virological index (i.e., the level of HBV DNA and HBsAg) in mouse serum was determined.
(71) 4.3 Analysis of Therapeutic Effects of Recombinant Proteins
(72) The detection results are shown in
(73) The results show that in the groups receiving immunotherapy with VLP, Anti-HBsAg antibodies were detected in mouse sera after immunization, and the level of HBV DNA and HBsAg decreased to different extents in mouse sera. By comparison, no Anti-HBsAg antibodies were generated in the sera of control mice (which were not immunized with VLP), and no decrease in the level of HBV DNA and HBsAg in sera was observed.
(74) These results show that all the 6 polypeptide carriers, constructed based on bat hepatitis B virus core protein, can be used to effectively present the epitope peptide (e.g., HBsAg-aa113-135) of HBsAg from human HBV, can form VLPs, and induce generation of high-titer anti-HBsAg antibodies in organisms, thereby inhibiting the level of HBV DNA and HBsAg (i.e., HBV DNA and HBsAg decreased significantly) in mice. In addition, the experimental data in
(75) Therefore, the experimental results in the Example show: (1) the polypeptide carriers of the invention can form VLPs, are suitable for presenting various target polypeptides, and can induce generation of high-titer antibodies against target polypeptides in organisms; (2) the polypeptide carriers of the invention are particularly suitable for presenting epitopes of human HBV (e.g., an epitope of HBsAg of human HBV), can induce generation of high-titer antibodies against HBsAg in organisms, and can clean or inhibit the level of HBV DNA and HBsAg in vivo, with an efficacy better than that of the polypeptide carrier constructed based on HBcAg of human HBV. Thus, the recombinant proteins presenting human HBV epitopes according to the invention are potential in treating HBV infection, and are particularly suitable for inducing effective, specific and therapeutic anti-HBV immunization.
Example 5. Preparation and Evaluation of Virus-Like Particles Presenting an Epitope of HBsAg from Different HBV Genotypes
(76) The HBsAg epitope (SEQ ID NO: 22) used in Example 2-4 was from HBV genotype B. In order to confirm the broad versatility of the polypeptide carrier of the invention for various HBV genotypes, the inventors also used RBHBcAg149 and TBHBcAg153 as exemplary polypeptide carriers, to construct the recombinant proteins presenting an epitope of HBsAg from different HBV genotypes (genotype A. C and D), and evaluated the ability of the constructed recombinant proteins to be assembled into virus-like particles, the immunogenicity of the virus-like particle produced, and the therapeutic effect thereof against HBV infection.
(77) 5.1 Construction of Expression Plasmids Encoding Recombinant Proteins Comprising a Target Polypeptide and a Polypeptide Carrier
(78) In the Example, in addition to the HBsAg epitope (from HBV genotype B, SEQ ID NO: 22) used in Example 2-4, the target polypeptide further includes the HBsAg epitope (amino acids from positions 113-135) from HBV genotype A, C and D, designated as: HBsAg-aa113-135-A, HBsAg-aa113-135-C and HBsAg-aa113-135-D. and their sequences (SEQ ID NO: 60-62) are shown in Table 4.
(79) TABLE-US-00005 TABLE 4 Sequences of amino acids from positions 113-135 of HBsAg protein fromHBV genotype A, C and D SEQ ID NO Name Sequence information 60 HBsAg-aa113-135-A STTTSTGPCKTCTTPAQGNSMFP 61 HBsAg-aa113-135-C TSTTSTGPCKTCTIPAQGTSMFP 62 HBsAg-aa113-135-D SSTTSTGPCRTCTTPAQGTSMYP
(80) The sense and antisense sequences (as shown in Table 5) coding said 3 target polypeptides were synthesized directly, and annealed, to obtain the gene fragments having cohesive end and encoding the target polypeptides.
(81) TABLE-US-00006 TABLE 5 Sense and antisense sequences coding 3 target polypeptides SEQ ID Primer NO: name Sequence information 63 HBsAg- GATCCTCTACCACCACCTCTACCGGTCCGTG aa113- CAAAACCTGCACCACCCCGGCTCAGGGTAAC 135-AF TCTATGTTCCCGG 64 HBsAg- AATTCCGGGAACATAGAGTTACCCTGAGCCG aa113- GGGTGGTGCAGGTTTTGCACGGACCGGTAGA 135-AR GGTGGTGGTAGAG 65 HBsAg- GATCCACCTCTACCACCTCTACCGGTCCGTG aa113- CAAAACCTGCACCATCCCGGCTCAGGGTACC 135-CF TCTATGTTCCCGG 66 HBsAg- AATTCCGGGAACATAGAGGTACCCTGAGCCG aa113- GGATGGTGCAGGTTTTGCACGGACCGGTAGA 135-CR GGTGGTAGAGGTG 67 HBsAg- GATCCTCTTCTACCACCTCTACCGGTCCGTG aa113- CCGTACCTGCACCACCCCGGCTCAGGGTACC 135-DF TCTATGTACCCGG 68 HBsAg- AATTCCGGGTACATAGAGGTACCCTGAGCCG aa113- GGGTGGTGCAGGTACGGCACGGACCGGTAGA 135-DR GGTGGTAGAAGAG
(82) As described in Example 2, the 3 gene fragments having cohesive end and encoding the target polypeptides as prepared above were ligated to linear vectors RBHBcAg149 and TBHBcAg153, respectively, so as to obtain the expression plasmids encoding the recombinant proteins (6 in total: RBHBcAg149-SEQ60, RBHBcAg149-SEQ61, RBHBcAg149-SEQ62, TBHBcAg153-SEQ60, TBHBcAg153-SEQ61, and TBHBcAg153-SEQ62). The amino acid sequences of the recombinant proteins encoded by the expression plasmids are shown in Table 6.
(83) TABLE-US-00007 TABLE 6 Amino acid sequences of 6 recombinant proteins SEQ. ID Recombinant NO: protein Sequence information 69 RBHBcAg149- MDIDPYKEFGASSQLISFLPEDFFPNLAELVET SEQ 60 TTALYEEELVGKEHCSPHHTALRSLLNCWGETV RLITWVRNSVEGGGGGSGGGGTGSSTTTSTGPC KTCTTPAQGNSMFPEFGGGGSGGGGSQDAIVQQ VQASVGLRMRQLMWFHLSCLTFGQPTVIEFLVS FGTWIRTPQAYRPPNAPILSTLPEHTIV 70 RBHBcAg149- MDIDPYKEFGASSQLISFLPEDFFPNLAELVET SEQ 61 TTALYEEELVGKEHCSPHHTALRSLLNCWGETV RLITWVRNSVEGGGGGSGGGGTGSTSTTSTGPC KTCTIPAQGTSMFPEFGGGGSGGGGSQDAIVQQ VQASVGLRMRQULMWFHLSCLTFGQPTVIEFLV SFGTWIRTPQAYRPPNAPILSTLPEHTIV 71 RBHBcAg149- MDIDPYKEFGASSQLISFLPEDFFPNLAELVET SEQ 62 TTALYEEELVGKEHCSPHHTALRSLLNCWGETV RLITWVRNSVEGGGGGSGGGGTGSSSTTSTGPC RTCTTPAQGTSMYPEFGGGGSGGGGSQDAIVQQ VQASVGLRMRQLMWFHLSCLTFGQPVIEFLVSF GTWIRTPQAYRPPNAPILSTLPEHTIV 72 TBHBc- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQ Ag153- LLESVNALYEDELTGPNHCSPHHTALRHLIMCG SEQ 60 VELRDFIDWMHEQGGGGGSGGGGTGSSTTTSTG PCKTCTTPAQGNSMFPEFGGGGSGGGGSDADAL LAGYLRSKYLKHITKAIWYHLSCLTIFGKQTVH EYLVSFGTWIRTPAAYRPVNAPILTTLPETSVI 73 TBHBcAg153- MENLERLDIYKEFGVSDVLVNSFLPDDFFPTLQ SEQ 61 QLLESVNALYEDELTGPNHCSPHHTALRHLIMC GVELRDFIDWMHEQGGGGGSGGGGTGSTSTTST GPCKTCTIPAQGTSMFPEFGGGGSGGGGSDADA LLAGYLRSKYLKHITKAIWYHLSCLTFGKQTVH EYLVSFGTWIRTPAAYRPVNAPILTTLPETSVI 74 TBHBcAg153- MENLERLDIYKEFGVSDVLVSFLPDDFFPTLQQ SEQ 62 ILESVNALYEDELTGPNHCSPHHTALRHLIMCG VELRDFIDWMHEQGGGGGSGGGGTGSSSTTSTG PCRTCTTPAQGTSMYPEFGGGGSGGGGSDADAL LAGYLRSKYLKHITKAIWYHLSCLTFGKQTVHE YLVSFGTWIRTPAAYRPVNAPILTTLPETSVI
(84) 5.2 Expression, Purification and Assembly of Recombinant Proteins
(85) As described in Example 2, by using the 6 expression plasmids constructed in the previous step, the recombinant proteins encoded by the expression plasmids were expressed and purified. Later, the VLPs formed by the recombinant proteins were observed by Transmission Electron Microscope (TEM).
(86)
(87) 5.3 Evaluation of Immunogenicity of Virus-Like Particles
(88) By using the method described in Example 3, the virus-like particles, formed by the 6 recombinant proteins as constructed above and the recombinant proteins RBHBcAg149-SEQ22 and TBHBcAg153-SEQ22 in Example 2, were evaluated for their immunogenicity. The experimental results are shown in
(89)
(90) 5.4 Evaluation on Anti-HBV Therapeutic Effects of Virus-Like Particles
(91) By using the method as described in Example 4, the virus-like particles formed by 4 recombinant proteins (SEQ ID NO: 36, 69, 70, and 71) were evaluated for the anti-HBV therapeutic effects. The experimental results are shown in
(92)
(93) These experimental results show that the polypeptide carriers of the invention (e.g., RBHBcAg149 and TBHBcAg153) can be used to effectively present epitope peptides (e.g., HBsAg-aa113-135) of HBsAg from human HBV of different genotypes (e.g., genotype A, B, C and D). The recombinant proteins, constructed based on the polypeptide carriers of the invention and epitope peptides of HBsAg, can form VLPs, and can induce the generation of high-titer anti-HBsAg antibodies in organisms, thereby inhibiting the HBsAg level (i.e., the HBsAg level decreased significantly) in mice. This indicates that the recombinant proteins comprising the polypeptide carriers of the invention and epitope peptides of HBsAg can be used to prevent and treat the infection by various HBV genotypes, and therefore can be used in the development of new anti-HBV vaccines and medicaments.
Example 6. Construction of Plasmids Encoding a Polypeptide Carrier Carrying a T Cell Epitope
(94) In this example, plasmids encoding a polypeptide carrier carrying a T cell epitope are constructed.
(95) 6.1 Preparation of Nucleotide Sequences Encoding a Polypeptide Carrier Carrying a T Cell Epitope
(96) Based on three bat-derived HBV core antigens (i.e., RBHBcAg protein, TBHBcAg protein, and HBHBcAg protein), the following polypeptide carriers were designed:
(97) RBHBcAg189-T3 carrier, which differs from RBHBcAg protein (SEQ ID NO: 1) in that: the amino acid residues from positions 78-81 of RBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid residues from positions 18-27 of RBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 87; the amino acid residues from positions 50-69 of RBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 88; and the amino acid residues from positions 120-140 of RBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 89. The amino acid sequence of RBHBcAg189-T3 carrier is set forth in SEQ ID NO: 75, the nucleotide sequence thereof is set forth in SEQ ID NO: 81.
(98) TBHBcAg188-T3 carrier, which differs from TBHBcAg protein (SEQ ID NO: 2) in that: the amino acid residues from positions 80-83 of TBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid residues from positions 18-27 of TBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 87; the amino acid residues from positions 54-73 of TBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 88; the amino acid residues from positions 124-144 of TBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 89. The amino acid sequence of TBHBcAg188-T3 carrier is set forth in SEQ ID NO: 76, the nucleotide sequence thereof is set forth in SEQ ID NO: 82.
(99) HBHBcAg189-T3 carrier, which differs from HBHBcAg protein (SEQ ID NO: 3) in that: the amino acid residues from positions 78-81 of HBHBcAg protein are substituted with a linker set forth in SEQ ID NO: 43; the amino acid residues from positions 18-27 of HBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 87; the amino acid residues from positions 50-69 of HBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 88; the amino acid residues from positions 120-140 of HBHBcAg protein are substituted with a sequence set forth in SEQ ID NO: 89. The amino acid sequence of HBHBcAg189-T3 carrier is set forth in SEQ ID NO: 77, the nucleotide sequence thereof is set forth in SEQ ID NO: 83.
(100) With respect to the nucleotide sequences of said 3 carriers, their whole gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd.
(101) 6.2 Preparation of Plasmids Encoding a Polypeptide Carrier
(102) By using the synthesized nucleotide sequences (e.g. RBHBcAg189-T3 carrier) as templates, and using the primers set forth in SEQ ID NOs: 45-47, the full-length gene and its truncate (i.e., a gene fragment truncated at C-terminus) of RBHBcAg189-T3 carrier were amplified by PCR. Two PCR products were obtained, i.e., the gene encoding RBHBcAg189-T3 carrier (SEQ ID NO: 81; the amino acid sequence encoded thereby is SEQ ID NO: 75), and the gene encoding RBHBcAg149-T3 carrier (SEQ ID NO: 84; the amino acid sequence encoded thereby is SEQ ID NO: 78).
(103) By a similar method, the following PCR products are obtained: the gene encoding TBHBcAg188-T3 carrier (SEQ ID NO: 82; the amino acid sequence encoded thereby is SEQ ID NO: 76); the gene encoding HBHBcAg189-T3 carrier (SEQ ID NO: 83; the amino acid sequence encoded thereby is SEQ ID NO: 77); the gene encoding TBHBcAg153-T3 carrier (SEQ ID NO: 85; the amino acid sequence encoded thereby is SEQ ID NO: 79); and the gene encoding HBHBcAg149-T3 carrier (SEQ ID NO: 86; the amino acid sequence encoded thereby is SEQ ID NO: 80).
(104) pTO-T7 vector (Luo Wenxin, Zhang Jun, Yang Haijie, et al., Construction and Application of an Escherichia coli High Effective Expression Vector with an Enhancer [J], Chinese Journal of Biotechnology, 2000, 16(5): 578-581) was subjected to double enzyme digestion by NdeI and HindIII, to obtain a linear vector. By Gibson assembly cloning method (New England Biolabs (UK) Ltd), the PCR products obtained were ligated to the linear vector, and transformed into DH5a competent bacteria. The transformed bacteria were spread on a plate and cultured, monoclonal colonies were then selected, and the plasmids were extracted and sequenced. It was confirmed by sequencing that 6 plasmids comprising the nucleotide sequences encoding the polypeptide carriers were obtained.
Example 7. Preparation of Recombinant Proteins
(105) In the Example, a nucleotide sequence encoding a target polypeptide was inserted into the plasmids constructed in Example 6, and a recombinant protein comprising the target polypeptide and the polypeptide carrier was obtained. The scheme of cloning solutions, in which recombinant proteins are constructed by inserting a target polypeptide (a target antigen peptide fragment) into RBHBcAg-T3 carrier, TBHBcAg-T3 carrier and HBHBcAg-T3 carrier of the invention, is shown in
(106) 7.1 Construction of Expression Plasmids Encoding a Recombinant Protein Comprising a Target Polypeptide and a Polypeptide Carrier
(107) In the Example, it was verified that the polypeptide carrier carrying a human T cell epitope of the invention could be used to present a target polypeptide. The exemplary target polypeptide used is HBsAg-aa113-135 (i.e., the amino acid residues from positions 113-135 of hepatitis B surface antigen (HBsAg) from human HBV, its amino acid sequence is set forth in SEQ ID NO: 22).
(108) A gene fragment having cohesive ends and encoding the target polypeptide HBsAg-aa113-135 is prepared as described in Example 2. The plasmids RBHBcAg189-T3 and RBHBcAg149-T3 obtained in Example 6 were subjected to double enzyme digestion by BamHI and EcoRI, to obtain 2 linear vectors. Then, the gene fragment having cohesive ends and encoding the target polypeptide, as prepared above, was ligated to each linear vector, respectively, to obtain the expression plasmids encoding the following recombinant proteins: RBHBcAg189-T3-SEQ22 (SEQ ID NO: 90) and RBHBcAg149-T3-SEQ22 (SEQ ID NO: 91).
(109) By a similar method, expression plasmids encoding the following recombinant proteins were obtained: TBHBcAg188-T3-SEQ22 (SEQ ID NO: 92), TBHBcAg153-T3-SEQ22 (SEQ ID NO: 93), HBHBcAg189-T3-SEQ22 (SEQ ID NO: 94) and HBHBcAg149-T3-SEQ22 (SEQ ID NO: 95).
(110) 7.2 Expression, Purification and Assembly of Recombinant Proteins
(111) The recombinant proteins encoded by the expression plasmids as prepared above were expressed and purified via the method described in Example 2.2, and used to assemble VLP.
(112)
(113) In addition, by the above method, it could be confirmed that the recombinant proteins TBHBcAg188-T3-SEQ22, TBHBcAg153-T3-SEQ22, HBHBcAg189-T3-SEQ22, and HBHBcAg149-T3-SEQ22 could also be assembled into well-formed virus-like particles.
(114) These results show that the polypeptide carriers carrying T cell epitopes constructed by the invention can be used for presenting a target polypeptide and can form VLP.
Example 8. Evaluation on Immunogenicity of Virus-Like Particles
(115) In the Example, the inventors verified the immunogenicity of the virus-like particles formed by the recombinant proteins prepared in Example 7. All such virus-like particles can induce generation of antibodies that specifically bind to target antigen in organisms.
(116) 8.1 Immunization of Mice
(117) BALB/C mice were immunized with the 2 virus-like particles (RBHBcAg189-T3-SEQ22 and RBHBcAg149-T3-SEQ22) prepared in Example 7 and the 2 virus-like particles (RBHBcAg189-SEQ22 and RBHBcAg149-SEQ22) prepared in Example 2, respectively. The immunization process was as following: the immunoadjuvant used was aluminum hydroxide adjuvant; the immunizing dose was 3 ug/dose; the immunization was performed by intramuscular injection at lateral thigh of hindlimb; the immune procedure was immunization once each at Week 0, 2, 3, 4, 5 and 6, 6 times in total.
(118) 8.2 Detection of Titer of Antibodies that Specifically Bind to the Target Antigen in Sera
(119) 8.2.1 Preparation of Reaction Plates
(120) The antigen for coating reaction plate was the target antigen corresponding to the target polypeptide SEQ22, i.e., human hepatitis B virus surface antigen recombinantly expressed in CHO cells (HBsAg, purchased from Beijing Wantai Biological Pharmacy).
(121) HBsAg protein was diluted with 50 mM CB buffer of pH9.6 (NaHCO.sub.3/Na.sub.2CO.sub.3 buffer, at a final concentration of 50 mM, pH=9.6), at a final concentration of 2 μg/mL, to obtain the coating solution. To each well of a 96-well ELISA plate, 100 μL coating solution was added, and the wells were coated at 2-8° C. for 16-24 h, and then further coated at 37° C. for 2 h. Later, PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) was used to wash wells once; and 200 μL blocking solution (20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer solution containing 20% bovine calf serum and 1% casein, pH=7.4) was then added to each well, and the wells were blocked at 37° C. for 2 h. The blocking solution was discarded. After that, the ELISA plate was dried, and packaged into an aluminum foil bag, which was stored at 2-8° C. for further use.
(122) 8.2.2 ELISA detection of Anti-HBsAg antibody titer in serum Collection of serum samples: blood was collected from the eye orbit of mice at Week 0, 2, 3, 4, 5, 6 and 7, the serum was separated and cryopreserved at −20° C., until detection.
(123) Sample dilution: a mouse serum was diluted with PBS solution containing 20% newborn bovine serum at 7 dilution gradients, i.e. 1:100, 1:500, 1:2500, 1:12500, 1:62500, 1:312500, and 1:1562500.
(124) ELISA detection: to each well of the coated ELISA plate, 100 μL diluted serum sample was added, and incubated at 37° C. for 30 min. The ELISA plate was then washed with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for five times. After washing, to each well of the ELISA plate, 100 μL GAM-HRP reaction solution was added, and incubated at 37° C. for 30 min. The ELISA plate was then washed with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for five times. After washing, to each well of the ELISA plate, 50 μL TMB color developing agent (provided by Beijing Wantai Biological Pharmacy) was added, and incubated at 37° C. for 15 min. After the incubation, to each well of the ELISA plate, 50 μL stop solution (provided by Beijing Wantai Biological Pharmacy) was added, and the OD450/630 value for each well was read by an ELISA instrument.
(125) Calculation of antibody titer: samples, the read values of which were within 0.2-2.0, were analyzed; a regression curve was plotted with the dilution fold and the read value, and the dilution fold of the sample, at which the read value was 2-fold of the background value, was calculated, and the dilution fold of the sample was used as the titer of the specific antibody in serum.
(126)
(127) By a similar method, it was verified that virus-like particles TBHBcAg188-T3-SEQ22, TBHBcAg153-T3-SEQ22, HBHBcAg189-T3-SEQ22, HBHBcAg149-T3-SEQ22 have good immunogenicity.
(128) The results show that the polypeptide carrier carrying a T cell epitope of the invention is suitable for presenting a target polypeptide, can form VLP; and the virus-like particle formed by the recombinant protein comprising the polypeptide carrier carrying a T cell epitope and the target polypeptide can induce generation of high-titer antibodies that specifically bind to the target polypeptide in an organism.
Example 9. Evaluation on Anti-HBV Therapeutic Effects of Virus-Like Particles Presenting the HBsAg Epitope (SEQ ID NO: 22)
(129) In the Example, the inventors evaluated the anti-HBV therapeutic effects of the virus-like particles presenting the same epitope peptide (SEQ ID NO: 22), as constructed based on different polypeptide carriers.
(130) 9.1 Immunization of Mice
(131) As described in Example 4, the virus-like particle RBHBcAg149-T3-SEQ22 prepared in Example 7 and the virus-like particle RBHBcAg149-SEQ22 prepared in Example 2 (2 virus-like particles presenting an epitope of HBsAg (SEQ ID NO: 22)) were evaluated for the anti-HBV therapeutic effects in an HBV transgenic mouse model.
(132) The immunization method was as followed: the immunoadjuvant used was aluminum hydroxide adjuvant; and the immunizing dose was 12 μg/dose; the immunization was performed by intramuscular injection at lateral thigh of hindlimb; the immune procedure was immunization once each at Week 0, 2, 3, 4, 5, and 6, 6 times in total.
(133) 9.2 Detection of Antibody Titer and Virological Index in Serum
(134) According to the method as described in Example 3.2, the Anti-HBsAg antibody titer in serum was determined, and the virological index (i.e., the level of HBV DNA and HBsAg) in mouse serum was determined.
(135) 9.3 Analysis of Therapeutic Effects of Recombinant Proteins
(136) The detection results are shown in
(137) The results show that in the groups receiving immunotherapy with the virus-like particle RBHBcAg149-T3-SEQ22 or the virus-like particle RBHBcAg149-SEQ22, after immunization, significant levels of Anti-HBsAg antibodies were detected in mouse sera, and the level of HBV DNA and HBsAg in mouse sera decreased significantly, and the efficacy of the two virus-like particles was comparable. In contrast, no Anti-HBsAg antibody was generated in the sera of control mice (which were not immunized with VLP), and no decrease in the level of HBV DNA and HBsAg in sera was observed.
(138) By a similar method, it is verified that virus-like particles RBHBcAg189-T3-SEQ22, TBHBcAg188-T3-SEQ22, TBHBcAg153-T3-SEQ22, HBHBcAg189-T3-SEQ22, and HBHBcAg149-T3-SEQ22 have good immunogenicity.
(139) These results show that all the polypeptide carriers carrying T cell epitopes, constructed based on bat hepatitis B virus core protein, can be used to effectively present the epitope peptide (e.g., HBsAg-aa113-135) of HBsAg from human HBV, can form VLPs, and induce generation of high-titer anti-HBsAg antibodies in hosts, thereby inhibiting the level of HBV DNA and HBsAg (i.e., HBV DNA and HBsAg decreased significantly) in mice. In addition, the experimental data in
(140) Therefore, the experimental results in the Example show: (1) the polypeptide carrier carrying a T cell epitope of the invention can form VLPs, is suitable for presenting a target polypeptide, and can induce generation of high-titer antibodies against the target polypeptide in an organism; (2) the polypeptide carrier carrying a T cell epitope of the invention is particularly suitable for presenting an epitope of human HBV (e.g., an epitope of HBsAg of human HBV), can induce generation of high-titer antibodies against HBsAg in an organism, and can clean or inhibit the level of HBV DNA and HBsAg in vivo, with an efficacy better than that of the polypeptide carrier constructed based on HBcAg of human HBV. Thus, the recombinant protein presenting a human HBV epitope according to the invention are potential in treating HBV infection, and is particularly suitable for inducing effective, specific and therapeutic anti-HBV immunization.
Example 10. Evaluation of Ability of a Virus-Like Particle Based on a Polypeptide Carrier Carrying a T Cell Epitope to Stimulate Immune Cells to Secrete IFNγ
(141) In the Example, we evaluated the ability of virus-like particles based on a polypeptide carrier carrying a T cell epitope to stimulate immune cells to secrete IFNγ. Briefly, the virus-like particle RBHBcAg149-T3-SEQ22 prepared in Example 7 and the virus-like particle RBHBcAg149-SEQ22 prepared in Example 2 were incubated with whole blood samples obtained from hepatitis B patients, respectively, and then the level of IFNγ in the whole blood samples was detected, so as to evaluate the ability of the virus-like particles to stimulate the immune cells in the whole blood samples of hepatitis B patients to secrete IFNγ.
(142) 10.1 Co-Culture of Virus-Like Particles and Whole Blood Samples
(143) Whole blood samples were collected from hepatitis B patients (26) and dispensed into three 1.5 mL EP tubes (500 μL per tube) and incubated respectively with virus-like particles RBHBcAg149-T3-SEQ22, RBHBcAg149-SEQ22 and PBS (used as a negative control, no toxoid) for 24 hours at 37° C. (at a final protein concentration of 2 μg/mL). After the incubation, the whole blood sample was centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected for the next test.
(144) 10.2 Detection of IFNγ
(145) According to the manufacturer's instructions, the MILLIPLEX MAP-Human Cell Signaling Portfolio, a human cytokine assay kit purchased from Millipore, was used to determine IFNγ levels in the supernatant samples. The results are shown in
(146)
(147) By a similar method, it could be verified that the virus-like particles RBHBcAg189-T3-SEQ22, TBHBcAg188-T3-SEQ22, TBHBcAg153-T3-SEQ22, HBHBcAg189-T3-SEQ22 and HBHBcAg149-T3-SEQ22 can also stimulate secretion of IFNγ by immune cells in whole blood samples obtained from hepatitis B patients.
(148) These results show that the polypeptide carrier carrying a human T cell epitope of the present invention and the recombinant protein/viral-like particle constructed based thereon can stimulate secretion of IFNγ by human immune cells, thus can enhance the immune response of human body to the recombinant protein/virus-like particle comprising the polypeptide carrier and the target polypeptide. The polypeptide carrier carrying a human T cell epitope of the present invention has a significant advantage in enhancing the response of human T cells.
Example 11. Preparation, Expression, Purification and Assembly of Recombinant Proteins Constructed Based on a Polypeptide Carrier without a Linker
(149) In the Example, the inventors used a polypeptide carrier without a linker to present a target polypeptide, so as to obtain a recombinant protein comprising the target polypeptide and the polypeptide carrier without the linker. Further, the inventors also studied the ability of the recombinant protein obtained to assemble into VLPs.
(150) Briefly, based on the expression plasmid encoding the recombinant protein RBHBcAg149-T3-SEQ22 obtained in Example 7, an expression plasmid expressing the recombinant protein RBHBcAg149n-T3-SEQ22 (having an amino acid sequence set forth in SEQ ID NO: 96) was constructed, wherein the recombinant protein RBHBcAg149n-T3-SEQ22 differs from the recombinant protein RBHBcAg149-T3-SEQ22 in that: the flexible linkers located at both ends of the target polypeptide SEQ22 are deleted. The construction method of the expression plasmid encoding the recombinant protein RBHBcAg149n-T3-SEQ22 is as follows.
(151) By using the expression plasmid encoding the recombinant protein RBHBcAg149-T3-SEQ22 as a template, PCR is performed using the first primer pair RBc149nF1 (SEQ ID NO: 97) and RBc149nR1 (SEQ ID NO: 98) to obtain the first amplification product; and PCR is performed using the second primer pair RBc149nF2 (SEQ ID NO: 99) and RBc149nR2 (SEQ ID NO: 100) to obtain the second amplification product. Subsequently, by using the first amplification product and the second amplification product together as templates, PCR is performed using primers RBc149nF1 (SEQ ID NO: 97) and RBc149nR2 (SEQ ID NO: 100) to obtain the third amplification product, i.e., a nucleic acid fragment encoding the recombinant protein RBHBcAg149n-T3-SEQ22.
(152) TABLE-US-00008 TABLE 7 primer sequences SEQ ID Primer NO: name sequences 97 RBc149nF1 AACTTTAAGAAGGAGATATACATATGGACATCG ACCCGTACAAAGAATTC 98 RBc149nR1 GAGVIGTGCAGGTTTTGCATGGTCCGGTGCTGG YMITGATGAACCVICAACAGAGTTAC 99 RBc149nF2 CAAAACCTGCACAACTCCTGCTCAAGGAACCTC TATGTTTCCCCAGGACGCTATCGTTCA 100 RBc149nR2 TGGTGCTCGAGTGCGGCCGCAAGCTTAGATAAC GGTGTGTTCCGGCAGGGTAG
(153) pTO-T7 vector was subjected to double enzyme digestion by NdeI and HindIII, to obtain a linear vector. By Gibson assembly cloning method (New England Biolabs (UK) Ltd), the third amplification product obtained was ligated to the linear vector, and transformed into ER2566 competent bacteria. The transformed bacteria were spread on a plate and cultured, monoclonal colonies were then selected, and the plasmids were extracted and sequenced. It was confirmed by sequencing that an expression plasmid with the nucleotide fragment encoding the recombinant protein RBHBcAg149n-T3-SEQ22 was obtained.
(154) Subsequently, the recombinant protein RBHBcAg149n-T3-SEQ22 was expressed and purified according to the method described in Section 2.2 of Example 2, and the purified recombinant protein was detected by SDS-PAGE and the VLPs formed by the recombinant protein was observed by the Transmission Electron Microscope. The experimental results were shown in
(155)
(156) Although the embodiments of the invention have been described in detail, a person skilled in the art would understand that according to all the disclosed teachings, details can be amended and modified, and these alterations all fall into the protection scope of the invention. The scope of the invention is defined by the attached claims and any equivalent thereof.