WO2020062930A1 - 一种重组病毒载体、包含其的免疫组合物以及用途 - Google Patents

一种重组病毒载体、包含其的免疫组合物以及用途 Download PDF

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WO2020062930A1
WO2020062930A1 PCT/CN2019/090877 CN2019090877W WO2020062930A1 WO 2020062930 A1 WO2020062930 A1 WO 2020062930A1 CN 2019090877 W CN2019090877 W CN 2019090877W WO 2020062930 A1 WO2020062930 A1 WO 2020062930A1
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antigen
seq
epitope
fusion peptide
tumor
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PCT/CN2019/090877
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French (fr)
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徐建青
黄杨
张晓燕
王璐
郜明泉
孙甲浩
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苏州工业园区唯可达生物科技有限公司
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Priority to US17/280,803 priority Critical patent/US20220002750A1/en
Publication of WO2020062930A1 publication Critical patent/WO2020062930A1/zh

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Definitions

  • the invention belongs to the fields of molecular biology and immunology. Specifically, the present invention relates to a recombinant viral vector comprising a polynucleotide encoding a CD4 helper T cell epitope fusion peptide, an immune composition comprising the same, and uses thereof.
  • T helper cells are T cells that play an important role in the immune system, especially in the adaptive immune system. They help the activity of other immune cells by releasing T-cell cytokines. These cells help to suppress or regulate the immune response. They are necessary for the conversion of B cell antibody classes, the activation and growth of cytotoxic T cells, and maximizing the bactericidal activity of phagocytic cells such as macrophages.
  • CD4 + T cells express the protein CD4, called CD4 + T cells.
  • CD4 + T cells are usually pre-defined as helper T cells in the immune system.
  • helper T cells such as CD40 (protein) and CD40L
  • CD4 + cells will help these cells through a combination of cell-to-cell interactions (such as CD40 (protein) and CD40L) and cytokines.
  • helper T cells can be seen in HIV, a virus that primarily infects CD4 + T cells.
  • HIV acquired immune deficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • lymphopenia a rare disease that result in the loss of CD4 + T cells or dysfunction. These diseases produce similar symptoms, many of which are fatal.
  • Antigenic epitope refers to a chemical group on the surface of an antigen that determines the specificity of an antigen.
  • An epitope can be recognized by the immune system (especially antibodies, B cells or T cells).
  • the region of an antibody that recognizes an epitope is called a “paratope” or “antibody determinant.”
  • an epitope usually refers to a part of a substance such as a foreign protein, as long as it can be recognized by the autoimmune system, it is also classified as an epitope.
  • Epitope of protein antigens are divided into two types of conformational epitopes and linear epitopes according to their structure and interaction with paratopes.
  • the conformational epitope consists of discontinuous parts in the amino acid sequence of the antigen, so the interaction between paratope and epitope is based on the three-dimensional characteristics and shape of the surface, or the tertiary structure of the antigen. Most epitopes belong to conformational epitopes.
  • a linear epitope consists of a continuous sequence of antigenic amino acids, and its interaction with the antigen is based on its primary structure.
  • T cell epitopes are mainly composed of short peptides composed of 8-17 amino acids, and appear on antigen-presenting cells (APCs). This epitope will be compatible with major tissues Complex (MHC) combines to form a complex, and binds to the corresponding T cell epitope receptor, thereby activating T cells and generating the corresponding cellular immune response (Shimonkevitz et al., 1984; Babbitt et al., 1985; Buus et et al., 1986; Townsend and Bodmer, 1989). There are mainly two types of MHC that bind to epitopes.
  • MHC major tissues Complex
  • the T cell antigen epitope presented by the major histocompatibility complex of class I is usually a polypeptide of 8 to 11 amino acids in length, while the major histocompatibility of class II is compatible.
  • the T-cell epitope presented by the sex complex is relatively long and consists of 13-17 amino acids.
  • Th epitopes refer to a class of T-cell epitopes in T-cell epitopes that are formed by binding to MHC molecules and which can be recognized by CD4 helper T-cell receptors.
  • the Th epitope mainly binds to molecules present on the surface of antigen-presenting cells (APCs) encoded by the class II genes of the major histocompatibility complex (MHC).
  • APCs antigen-presenting cells
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • helper T-cell epitopes for peptide immunogens is the carrier protein for covalent coupling of peptides, but this coupling process can introduce other problems, such as the modification of epitopes and the targeting of peptides during the coupling process.
  • Induction of antibodies against vectors at the expense of antibodies Schott, MP, Leclerc, C. Jolivet, M. Audibert, F. Chedid, L. Carrier-induced epitopic suppression, a major issue for futuresynthetic vaccinations. J Immunol. 1985, 135,2319-2322; DiJohn, D., Torrese, JRMurillo, J. Herrington, DAetal.Effect of the carrier with the carrier on the response to the vacuum.
  • immunogens must contain helper T cell epitopes, in addition to epitopes to be recognized by surface Ig or receptors present on cytotoxic T cells. It should be appreciated that these types of epitopes can be very different. For B-cell epitopes, conformation is important because B-cell receptors bind directly to the native immunogen. In contrast, epitopes recognized by T cells are not dependent on the conformational integrity of the epitopes, and are composed of a short sequence of about 9 amino acids against CTL and a slightly longer sequence against helper T cells (less restrictive in length) composition.
  • these epitopes can be housed in the binding clefts of class I or class II molecules, respectively, and then the complex can bind to T cell receptors.
  • the binding sites of Class II molecules are open at both ends, allowing for reporting to as short as 8 amino acid residues (Fahrer, AM, Geysen, HM, White, DO, Jackson, DC and Brown, LEAnalysis of the requirements class II-restricted T-cell recognition, of which single-term determinants are disclosed, which is considerable, in the T-cell response, and degeneracy of peptide binding to HED Immunol.
  • Th epitopes can stimulate and activate helper T cells, and accordingly, promote the activation of CD8 T cells and B cells, and ultimately improve the immune response.
  • the Th epitope in addition to activating the immune response against itself, the Th epitope can effectively help the immune response of other antigens or epitopes associated with it. Therefore, a heterologous strong Th epitope can be fused with the target immunogen, and the immunogenicity of the target immunogen can be improved accordingly.
  • PADRE pan HLA DR-binding Epitope
  • Th epitope P2 derived from Tetanus toxin is also commonly used to couple with the immunogen of interest to improve immunogenicity (Panina-BordignonPet et al., Eur J Immunol, 1989, 19: 2237 -42; La Rosa, Corinna et al., The Journal of Infectious Diseases, 2012, 205: 1294-304).
  • the Th epitope used to increase immunogenicity is usually heterologous, in other words, the vaccine subject will not have a high level of immune response against the Th epitope itself. Therefore, when vaccinating a subject with the strong Th epitope described above, it is likely that the immune system of the vaccine subject is first exposed to such Th epitopes, and the recipient immune system is activated to target such Th epitopes and purposes
  • the epitope of the immunogen is basically synchronous.
  • the generation time and number of T cells targeting such Th epitopes are similar to the target immunogen. In this case, the effect on helping the target immunogen will be limited. Especially for weakly immunogenic tumor antigens, the auxiliary role of such Th epitopes is more difficult to exert.
  • Th epitopes can activate tumor antigens, the level of cellular immune responses they stimulate is still low and cannot meet the needs of tumor vaccines (Ghaffari-Nazari H et al., PLoS ONE, 2015, 10 (11 ): e0142563).
  • Th epitope strategy is needed to improve the immunogenicity of the target immunogen, especially some weak immunogens, such as tumor antigens.
  • the object of the present invention is to provide a CD4 helper T cell epitope fusion peptide, and the epitope fusion peptide can improve the immunogenicity of the target immunogen.
  • the present invention utilizes strong Th epitopes derived from cytomegalovirus (CMV) and influenza virus (Influvirus, Flu) to obtain epitope fusion peptides to improve the immunogenicity of the target immunogen.
  • CMV cytomegalovirus
  • Influenvirus, Flu influenza virus
  • Epitope fusion peptide refers to a peptide formed by joining several epitopes together.
  • target immunogen refers to an immunogen used for achieving an immune response, including an antigen having an immunological activity, such as an antigen, and preferably a protein.
  • Another object of the present invention is to provide a fusion protein of the epitope fusion peptide and the immunogen of interest.
  • the present invention provides a CD4 helper T cell epitope fusion peptide, which comprises a cytomegalovirus epitope and / or an influenza virus epitope.
  • the present invention also provides a recombinant viral vector comprising a polynucleotide encoding a CD4 helper T cell epitope fusion peptide, and the CD4 helper T cell epitope fusion peptide comprises a cytomegalovirus epitope and / Or influenza virus epitopes.
  • the epitope fusion peptide comprises one or more selected from the cytomegalovirus epitope shown in SEQ ID NO: 1-10, and / or selected from SEQ ID NO: One or more of the influenza virus epitopes shown in 11-23.
  • the epitope fusion peptide is selected from one or more of the cytomegalovirus epitopes shown in SEQ ID NO: 1-10, and / or selected from SEQ ID NO: One or more of the influenza virus epitopes shown in 11-23.
  • the epitope fusion peptide is composed of 5 or 10 cytomegalovirus epitopes and / or 8 or 13 influenza virus epitopes, for example, an epitope fusion peptide shown in SEQ ID NO: 34 or 44 .
  • the epitope fusion peptide consists of 13 influenza virus epitopes, such as the epitope fusion peptide shown in SEQ ID NO: 48 or 60.
  • the epitope fusion peptide induces a humoral or cellular immune response.
  • the recombinant virus is a vaccinia virus, preferably a replicating vaccinia virus vector, such as a vaccinia virus Tiantan strain, such as the 752-1 strain, or a non-replicating vaccinia virus vector, such as a modified vaccinia virus Ankara strain (Modified Vaccinia Ankara strain). , MVA).
  • a replicating vaccinia virus vector such as a vaccinia virus Tiantan strain, such as the 752-1 strain
  • a non-replicating vaccinia virus vector such as a modified vaccinia virus Ankara strain (Modified Vaccinia Ankara strain). , MVA).
  • the invention also provides a fusion protein of the epitope fusion peptide and the immunogen of interest.
  • the invention also provides a polynucleotide encoding the epitope fusion peptide and / or the fusion protein.
  • the immunogen of interest is any one or more immunogens.
  • the immunogen of interest is a peptide, an antigen, a hapten, a carbohydrate, a protein, a nucleic acid, an allergen, a virus or a part of a virus, a bacterium, a parasite, or another intact microorganism.
  • the antigen is a tumor antigen or an infection-associated antigen.
  • the tumor antigen is selected from one or more of a lung cancer antigen, a testicular cancer antigen or a melanoma antigen, a liver cancer antigen, a breast cancer antigen, or a prostate cancer antigen.
  • the tumor antigen is selected from one or more of a LAGE antigen, a MAGE antigen, or a NY-ESO-1 antigen.
  • the LAGE antigen is LAGE-1 and the MAGE antigen is MAGE-A3.
  • the tumor antigen comprises LAGE-1, MAGE-A3 and NY-ESO-1.
  • the amino acid sequence of LAGE-1 is shown in SEQ ID NO: 24, the amino acid sequence of MAGE-A3 is shown in SEQ ID NO: 25, and the amino acid sequence of NY-ESO-1 is shown in SEQ ID It is shown in NO: 26; still further preferably, the tumor antigen comprises LAGE-1, MAGE-A3 and NY-ESO-1.
  • the infection-associated antigen is selected from one or more of an HIV antigen, an influenza virus antigen, or an HBV antigen.
  • the fusion protein is as shown in one of SEQ ID NOs: 55-58, 62.
  • the gene encoding the fusion protein is shown in SEQ ID NO: 63.
  • Another object of the present invention is to provide an immune composition comprising a therapeutically effective amount of an epitope fusion peptide, a fusion protein, a polynucleotide and / or a recombinant viral vector according to the present invention, and a pharmaceutically acceptable Accepted vectors.
  • the immune composition is a vaccine.
  • Another object of the present invention is to provide a kit comprising an epitope fusion peptide, a fusion protein, a polynucleotide, a recombinant viral vector and / or an immune composition according to the present invention, and instructions for its use.
  • the present invention also provides the use of an epitope fusion peptide, fusion protein, polynucleotide, recombinant viral vector and / or immune composition according to the present invention in the preparation of a medicament or vaccine that improves the immunogenicity of the immunogen of interest.
  • the present invention also provides a method for improving the immunogenicity of an immunogen of interest using an epitope fusion peptide according to the present invention, which comprises a CD4 helper T cell epitope that has a strong immune response in a vaccine subject or a population A fusion protein formed by fusion with a target immunogen.
  • the method is specifically:
  • the epitope of one or more CD4 helper T cells is selected, and the complex formed by binding the epitope to the MHC molecule can be recognized by the CD4 helper T cell receptor, and before the vaccination, the body of the vaccine subject There is already a T-cell immune response against at least one of the epitopes;
  • the above epitope is fused to make an epitope fusion peptide, the epitope fusion peptide is fused with a target immunogen to make a fusion protein, the fusion protein is expressed and a vaccine is made, and the expression vector form may be a DNA vaccine
  • the vector form may be a protein vaccine vector form or a viral vaccine vector form;
  • the above vaccine is vaccinated to a vaccine subject, and an appropriate adjuvant can be selected during the vaccination, such as incomplete Freund's adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, and the like.
  • an appropriate adjuvant can be selected during the vaccination, such as incomplete Freund's adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, and the like.
  • step (1) of the method of use further includes the step of checking the MHC phenotype of the vaccine subject.
  • examining the MHC phenotype of the vaccine subject includes examining the MHC class II gene subtype of the vaccine subject.
  • the invention also provides a method of treating or preventing a condition in a subject in need thereof, comprising administering a therapeutically effective amount of an epitope fusion peptide, an immune composition, a polynucleotide, and / or a recombinant viral vector of the invention.
  • the condition is selected from one or more of a malignancy, a bacterium, and a viral infection.
  • the malignant tumor is breast cancer or colon cancer.
  • the infection is a chronic infection.
  • a DNA vaccine vector is used for primary immunization
  • a protein vaccine vector is used for enhanced immunity. More preferably, a pVKD1.0-CI-LMNB DNA vaccine is used for primary immunization, and LMNB-I13 protein is used for enhanced immunity.
  • the epitope fusion peptide provided by the invention can greatly improve the cellular immune response level of the target immunogen, especially the weak immunogen, and overcome the immune tolerance of the immune system to the antigen, especially the immune tolerance to tumor antigens or infection-related antigens. It is an effective means for enhancing the effectiveness of vaccines.
  • Figure 1 and Figure 2 are the plasmid map and double-enzyme identification map of DNA vaccine vector pVKD1.0-hLMN with LAGE-1, MAGE-A3 and NY-ESO-1 antigen coding sequences, respectively.
  • Figure 3 and Figure 4 are plasmid maps and double-enzyme identification of DNA vaccine vector pVKD1.0-hLMN-CTB with LAGE-1, MAGE-A3 and NY-ESO-1 antigens and coding sequences for the cholera toxin B subunit, respectively Illustration.
  • Figures 5 and 6 are plasmid maps and double-enzyme identification maps of DNA vaccine vector pVKD1.0-CI with CMV and influenza virus CD4 epitope coding sequences, respectively.
  • Figures 7 and 8 are DNA vaccine vectors pVKD1.0- with the LAGE-1, MAGE-A3 and NY-ESO-1 antigens and the cholera toxin B subunit, and derived from the CMV and influenza virus CD4 epitope coding sequences.
  • Figures 9 and 10 are the plasmid map and double-enzyme identification map of the prokaryotic vector pET-30a (+)-LMN with LAGE-1, MAGE-A3 and NY-ESO-1 antigen coding sequences, respectively.
  • Figures 11 and 12 are plasmid maps and dual enzymes of the prokaryotic vector pET-30a (+)-LMN-CTB with the LAGE-1, MAGE-A3 and NY-ESO-1 antigens and the coding sequence of the cholera toxin B subunit, respectively Cut identification map.
  • Figures 13 and 14 are the plasmid maps and double-enzyme identification maps of the prokaryotic vector pET-30a (+)-CMV Th with a CMV epitope coding sequence, respectively.
  • Figure 15 and Figure 16 are prokaryotic vectors pET-30a (+)-CMV10-LMNB with CMV epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens and coding sequences for the cholera toxin B subunit, respectively Plasmid map and double enzyme digestion map.
  • Figures 17 and 18 are plasmid maps and double-enzyme identification maps of the prokaryotic vector pET-30a (+)-CMV Th with a coding sequence derived from influenza virus, respectively.
  • Figures 19 and 20 are prokaryotic vectors pET-30a (+)-Influ8- Plasmid map and double enzyme digestion map of LMNB.
  • Figure 21 and Figure 22 are the prokaryotic vector pET-30a (+)-Influ13- with the influenza virus-derived epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and the coding sequence of the cholera toxin B subunit, respectively Plasmid map and double enzyme digestion map of LMNB.
  • Figure 23 shows the results of the cellular immune response test in animal immunity experiments.
  • Figures 24 and 25 are prokaryotic vectors pET-30a (+)-CMV5-LMNB with CMV epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens and coding sequences for the cholera toxin B subunit, respectively Plasmid map and double enzyme digestion map.
  • FIG. 26 is a result of detection of a cellular immune response in an animal immunity experiment in Example 12.
  • FIG. 26 is a result of detection of a cellular immune response in an animal immunity experiment in Example 12.
  • Figure 27 shows the tumor growth of mice in Example 13.
  • Figure 28 and Figure 29 are the tumor-free survival status and overall survival status of the mice in Example 13, respectively.
  • Figure 30 shows the tumor growth of mice in each treatment group in a 4T1-hNY-ESO-1 mouse tumor model.
  • Figure 31 shows the tumor growth of mice in each treatment group in the CT26-hLAGE-1 mouse tumor model.
  • Figure 32 and Figure 33 are plasmid maps and double clones of the shuttle vector pSC65-LMNB-I13 with the LAGE-1, MAGE-A3 and NY-ESO-1 antigen coding sequences and 13 influenza epitope coding sequences (I13), respectively. Enzymatic digestion map.
  • FIG. 34 shows the tumor growth of each group of mice in the CT26-MAGE-A3 mouse tumor model in Example 19.
  • FIG. 35 shows the overall survival status of mice in each group in the CT26-MAGE-A3 mouse tumor model in Example 19.
  • FIG. 36 shows the tumor growth of each group of mice in the CT26-LAGE-1 mouse tumor model in Example 20.
  • FIG. 37 shows the overall survival status of mice in each group in the CT26-LAGE-1 mouse tumor model in Example 20.
  • the amino acid sequences of LAGE-1, MAGE-A3 and NY-ESO-1 are shown in SEQ ID NO: 24-26, respectively.
  • the above amino acid sequence of the antigen was optimized into a mammalian codon usage preference nucleotide sequence through online codon optimization software (http://www.jcat.de/), as shown in SEQ ID NO: 27-29, respectively.
  • After synthesis by Shanghai Jereh Biotechnology Co., Ltd. it was cloned into the multi-cloning site Sal I and BamH I on the DNA vaccine vector pVKD1.0 (provided by Suzhou Industrial Park Vekida Biotechnology Co., Ltd.) by methods well known in the art.
  • a DNA vaccine vector pVKD1.0-hLMN (plasmid map shown in Figure 1) was constructed that can express the fusion protein antigen.
  • the restriction enzymes Sal I and BamH I were used to identify the vector pVKD1.0-hLMN (the digestion system is shown in Table 1).
  • the digestion verification map is shown in Figure 2.
  • Enzyme digestion system volume Plasmid pVKD1.0-hLMN 3 ⁇ L, about 1 ⁇ g Sal I (Treasure Bio, Cat. No. 1080A) 1 ⁇ L BamH I (BaoHing, article number 1010A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • Cholera toxin subunit B (CTB) amino acid sequence SEQ ID NO: 30
  • mammalian codon optimized sequence SEQ ID ID: 31
  • pVKD1.0-CTB Cholera toxin subunit B amino acid sequence
  • pVKD1.0-CTB design primers (Table 2), amplify the CTB gene fragment by PCR, then gel recover the corresponding fragment, and insert the CTB fragment into the corresponding position of the linearization vector pVKD1.0-hLMN by homologous recombination.
  • a DNA vaccine vector pVKD1.0-hLMN-CTB was constructed (the plasmid map is shown in Figure 3), which was identified by sequencing and stored in the library.
  • the restriction enzymes Sal I and BamH I were used to identify the vector pVKD1.0-hLMN-CTB (the digestion system is shown in Table 3).
  • the digestion verification map is shown in Figure 4.
  • Table 3 Identification system for digestion of plasmid pVKD1.0-hLMN-CTB (digestion at 37 ° C for 2 hours)
  • Enzyme digestion system volume Plasmid pVKD1.0-hLMN-CTB 3 ⁇ L, about 1 ⁇ g Sal I (Treasure Bio, Cat. No. 1080A) 1 ⁇ L BamH I (BaoHing, article number 1010A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • CMV Cytomegalovirus
  • IEDB immune epitope database
  • the strong Th epitopes of CMV include pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112, and pp65-104; influenza viruses Strong Th epitopes include HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127, and M1-210.
  • the epitope selected in Table 4 covers most subtypes of MHC class II molecules in the population, and also covers mouse MHC class II molecules. Then connect the selected epitopes pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221 in series, An epitope fusion peptide of CMV virus and influenza virus is formed.
  • the amino acid sequence is shown in SEQ ID NO: 34.
  • the nucleic acid sequence is shown in SEQ ID NO: 35. Send to Suzhou Hongxun Biotechnology Co., Ltd.
  • the vector pVKD1.0-CI (plasmid map is shown in Figure 5) was identified by sequencing and stored in the library.
  • the restriction enzymes Pst I and Bgl II were used to identify the vector pVKD1.0-CI (the digestion system is shown in Table 5).
  • the digestion verification spectrum is shown in Figure 6.
  • Enzyme digestion system volume Plasmid pVKD1.0-CI 3 ⁇ L, about 1 ⁇ g Pst I (Bao Biology, article number 1073A) 1 ⁇ L Bgl II (Bo Bio, Cat. No. 1021A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • Table 7 Identification system for digestion of plasmid pVKD1.0-CI-LMNB (digestion at 37 ° C for 2 hours)
  • Enzyme digestion system volume Plasmid pVKD1.0-CI-LMNB 3 ⁇ L, about 1 ⁇ g Bam HI (Bao Biological, article number 1010A) 1 ⁇ L EcoR V (Bo Bio, Cat. No. 1042A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • the amino acid sequences of LAGE-1, MAGE-A3 and NY-ESO-1 are shown in SEQ ID NO: 24-26, respectively.
  • the on-line codon optimization software http://www.jcat.de/
  • prokaryotic expression construct pET-30a (+)-LMN was constructed (plasma map is shown in Figure 9).
  • the restriction enzymes Nco I and Xho I were used to identify the vector pET-30a (+)-LMN (the digestion system is shown in Table 8).
  • Table 8 Identification system for digestion of plasmid pET-30a (+)-LMN (digestion at 37 ° C overnight)
  • Enzyme digestion system volume Plasmid pET-30a (+)-LMN 3 ⁇ L, about 1 ⁇ g Nco I (Bo Bio, article number 1160A) 1 ⁇ L Xho I (Bao Biology, article number 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • the cholera toxin B subunit CTB amino acid sequence (SEQ ID NO: 30) and its prokaryotic codon optimized nucleic acid sequence (SEQ ID NO: 41) were provided by Suzhou Industrial Park Vekida Biotechnology Co., Ltd. Design primers (Table 9), use pET-30a (+)-CTB (Suzhou Industrial Park Vekida Biotech Co., Ltd.) as a template, and amplify the nucleic acid fragment containing the CTB coding sequence by PCR method. For details, see ExTaq enzyme. (Po Bio, Cat. No. RR001B) Reagent Instructions.
  • This nucleic acid fragment was then inserted into the pET-30a (+)-LMN vector by homologous recombination to construct a pET-30a (+)-LMN-CTB vector (the plasmid map is shown in Figure 11).
  • Library. The restriction enzymes Nco I and Xho I were used to identify the vector pET-30a (+)-LMN-CTB (the digestion system is shown in Table 10).
  • Enzyme digestion system volume Plasmid pET-30a (+)-LMN-CTB 3 ⁇ L, about 1 ⁇ g Nco I (Bo Bio, article number 1160A) 1 ⁇ L Xho I (Bao Biology, article number 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L
  • Th epitopes from Table 4 from CMV pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104, connected in tandem, the amino acid sequence is as shown in SEQ ID NO: 44, where a sequence of "EFELRRQ" in SEQ ID NO: 44 is caused by the introduction of a restriction site, It is a commonly used technology for fusion construction.
  • the Th epitope amino acid sequence was optimized into the E. coli codon usage preference nucleotide sequence (SEQ ID NO: 45) by online codon optimization software (http://www.jcat.de/).
  • CMV Th1 contains 5 CMV Th epitopes, which are made up of pp65-11, pp65-71, pp65-92, pp65-123 and pp65-128, and CMV Th2 contains 5 CMV.
  • Th epitope consisting of pp65-57, pp65-62, pp65-30, pp65-112, and pp65-104.
  • Three restriction enzymes including EcoR I, Sac I, and Sal I were introduced between CMV Th1 and CMV Th2. Site.
  • Table 11 Plasmid pET-30a (+)-CMV Th restriction enzyme identification system (digestion at 37 ° C overnight)
  • Enzyme digestion system volume Plasmid pET-30a (+)-CMV Th 3 ⁇ L, about 1 ⁇ g Mlu I (Bo Bio, article number 1071A) 1 ⁇ L Xho I (Bao Biology, article number 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • the restriction endonucleases BamH I and Xho I were used to identify the vector pET-30a (+)-CMV10-LMNB (the restriction system is shown in Table 13).
  • the digestion verification map is shown in Figure 16.
  • pET-30a (+)-CMV10-LMNB contains CMV Th1 and CMV Th2 fragments, that is, the vector contains all 10 CMV Th epitopes in Table 4.
  • the epitopes are pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112, and pp65-104.
  • Primer sequence 6F (SEQ ID NO: 46) GCGCGGCCGCGACGACAAGGCCATGGCT 6R (SEQ ID NO: 47) GCCTCGAGGTTAGCCATAGAGATAGC
  • Enzyme digestion system volume Plasmid pET-30a (+)-CMV10-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (BaoHing, article number 1010A) 1 ⁇ L Xho I (Bao Biology, article number 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • influenza epitopes HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127 and M1-210 derived from influenza virus were selected from Table 4. It is connected in tandem, and its amino acid sequence is shown in SEQ ID NO: 48.
  • the online codon optimization software http://www.jcat.de/ was used to optimize the amino acid sequence containing the Th epitope of influenza virus into the nucleotide sequence of E. coli codon usage preference (SEQ ID NO: 49). After being synthesized by Shanghai Jereh Biotechnology Co., Ltd., it was inserted into the multi-cloning sites Nco I and Xho I of the prokaryotic expression vector pET-30a (+) (Novagen, Cat. No. 69909) by molecular biology methods well known in the art. At the same time, a prokaryotic expression construct pET-30a (+)-Influ Th (the plasmid map is shown in Figure 17) was constructed, which can express the fusion protein antigen. The restriction enzymes Nco I and Xho I were used to identify the vector pET-30a (+)-Influ Th (the digestion system is shown in Table 14). The digestion verification map is shown in Figure 18.
  • Influ Th1 contains 8 epitopes of influenza virus, which are formed by tandem HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95 and NP221, and Influ Th2 contains 5 influenzas.
  • the Th epitope of the virus is composed of HA434, HA440, NP324, M1-127 and M1-210.
  • Influ Th1 and Influ Th2 introduce 3 restriction sites such as EcoR I, Sac I and Sal I.
  • the pET-30a (+)-Influ8-LMNB vector contains the Influ Th1 fragment, which includes HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 in Table 4. Eight epitopes of the influenza virus.
  • Table 16 Identification system for digestion of plasmid pET-30a (+)-Influ8-LMNB (digestion at 37 ° C overnight)
  • Enzyme digestion system volume Plasmid pET-30a (+)-Influ8-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (BaoHing, article number 1010A) 1 ⁇ L Xho I (Bao Biology, article number 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • the pET-30a (+)-Influ13-LMNB vector contains Influ Th1 and Influ Th2 fragments, that is, HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and Eight influenza virus Th epitopes including NP221 and five influenza virus Th epitopes including HA434, HA440, NP324, M1-127 and M1-210 in Table 4.
  • This vector includes a total of all 13 influenza virus Th epitopes in Table 4.
  • Primer sequence 7F2 (SEQ ID NO: 52) GCCTCGAGGTTAGCCATAGAGATAGCA 7R2 (SEQ ID NO: 53) GCGCGGCCGCGACGACAAGGCCATGGCTATG
  • Enzyme digestion system volume Plasmid pET-30a (+)-Influ13-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (BaoHing, article number 1010A) 1 ⁇ L Xho I (Bao Biology, article number 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • the prokaryotic expression vector pET-30a (+)-LMN constructed in Example 4 the prokaryotic expression vector pET-30a (+)-LMN-CTB constructed in Example 5, and the prokaryotic expression vector pET constructed in Example 6, respectively -30a (+)-CMV5-LMNB and pET-30a (+)-CMV10-LMNB, the prokaryotic expression vectors pET-30a (+)-Influ8-LMNB and pET-30a (+)-Influ13- LMNB transformed into BL21 (DE3) competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No.
  • the BCA method (Biyuntian Biotechnology Research Institute, article number P0009) detected that the concentration of the prepared recombinant protein was 1 mg / mL.
  • the detection method refer to the instruction manual of the detection kit.
  • the gel method Xiamen ⁇ Reagent Experiment Factory Co., Ltd., article number G011000
  • the endotoxin content of the prepared recombinant protein was ⁇ 1EU / mg, which meets the requirements of animal experiments.
  • the detection method refer to the instructions of the ⁇ reagent.
  • DNA vaccine vector pVKD1.0 was provided by Suzhou Industrial Park Vickers.
  • the DNA vaccine pVKD1.0-NP expression derived from strain A / Shanghai / 02/2013 (H7N9)
  • influenza antigen NP NCBI reference sequence: YP_009118476 .1
  • the protein vaccine VP1 Enterovirus 71 type VP1 protein, see Chinese patent application 201310088364.5
  • Suzhou Industrial Park Vekuda Co is provided by Suzhou Industrial Park Vekuda Co.
  • mice Sixteen 6-8 week-old female BAL B / c mice were purchased from the Animal Experiment Center of Soochow University and kept in the SPF animal room of Animal Experiment Centre of Soochow University. The experimental animal grouping and vaccination plan are shown in Table 20. All DNA vaccines were injected into the calf anterior tibial muscle, 100 ⁇ g / head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously on the back, 10 ⁇ g / head. Two weeks after the last immunization was completed, mice were sacrificed, serum and spleen cells were collected, and subjected to an enzyme-linked immunospot assay (ELISPOT) and an enzyme-linked immunoassay (ELISA), respectively.
  • ELISPOT enzyme-linked immunospot assay
  • ELISA enzyme-linked immunoassay
  • the mouse IFN- ⁇ ELISPOT kit was purchased from BD Company (Cat. No .: 551083).
  • the stimulating peptide was NY-ESO-1 41 # peptide (WITQCFLPVFLAQPP), which was synthesized by Shanghai Ke Peptide Biotechnology Co., Ltd.
  • the final stimulating concentration was 10 ⁇ g / mL.
  • the positive stimuli phorbol-12-myristate-13-acetate (PMA) and inomysin were purchased from Sigma Company, USA.
  • the method of ELISA is well known to those skilled in the art and is briefly described below.
  • the 96-well microtiter plate was purchased from Jianghai Glass Instrument General Factory. Both the restructured LMN and NY-ESO-1 were provided by Suzhou Industrial Park Vickers Biotechnology Co., Ltd.
  • the protein was coated with NaHCO 3 buffer (pH 9.6), overnight at 4 ° C, and the coating concentration was 10 ⁇ g / mL.
  • 3,3,5,5-tetramethylbenzidine (3,3,5,5-tetraamthyl benzidine, TMB) substrate was developed, 37 ° C, 15 minutes, and terminated with 2M dilute sulfuric acid using a microplate reader (USA Thermo) read the absorbance (A) value at a wavelength of 450 nm.
  • a positive value greater than a negative control A value of 2.1 was determined, and the reciprocal of the highest dilution in the positive value was defined as the serum antibody titer. When the titer is less than the initial dilution of 1: 100, the titer is defined as 50.
  • the results of the cellular immune response test are shown in FIG. 23.
  • the primed pVKD1.0-CI-LMNB DNA vaccine, the LMNB-I13 protein boost ie, group D in Example 8
  • had the best immune effect which was significantly higher than its parallel control (group B) and LMNB-I8 boost Group (Group C)).
  • the level of cellular immune response in the LMNB-I13 protein-enhanced group was increased by nearly three times compared with the parallel control group (group B). It is shown that the 13 th epitopes (group D) containing influenza virus can greatly improve the level of cellular immune response of weak immunogens.
  • the restriction enzymes BamH I and Xho I were used to identify the vector pET-30a (+)-CMV5-LMNB (the digestion system is shown in Table 22).
  • the digestion verification map is shown in Figure 25.
  • pET-30a (+)-CMV5-LMNB contains the CMV Th1 fragment, that is, the vector contains the first 5 CMV Th epitopes in Table 4. These epitopes are pp65-11, pp65-71, pp65-92, pp65-123, and pp65-128.
  • Primer sequence 7F1 (SEQ ID NO: 50) GCGCGGCCGCGTTAGCCATAGAGATAGC 7R1 (SEQ ID NO: 51) GCGTCGACAAGACGACAAGGCCATGGCTATGC
  • Table 22 pET-30a (+)-CMV10-LMNB digestion identification system (cut at 37 ° C overnight)
  • Enzyme digestion system volume Plasmid pET-30a (+)-CMV10-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (BaoHing, article number 1010A) 1 ⁇ L Xho I (Bao Biology, article number 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Fill up to 10 ⁇ L
  • the prokaryotic expression vector pET-30a (+)-CMV5-LMNB constructed in Example 10 was transformed into BL21 (DE3) competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd., article number CB105, For the transformation method, please refer to the instruction manual of competent cells).
  • BL21 DE3 competent cells
  • LMNB-C5 the amino acid sequence is shown in SEQ ID NO: 57
  • pET System Manual (TB055 8th Edition 02/99, Novagen). , Store at -80 ° C.
  • the BCA method (Biyuntian Biotechnology Research Institute, article number P0009) detected that the concentration of the prepared recombinant protein was 1 mg / mL.
  • the detection method refer to the instruction manual of the detection kit.
  • the gel method Xiamen ⁇ Reagent Experiment Factory Co., Ltd., article number G011000
  • the endotoxin content of the prepared recombinant protein was ⁇ 1EU / mg, which meets the requirements of animal experiments.
  • the detection method refer to the instructions of the ⁇ reagent.
  • Vaccine information is shown in Table 19.
  • the DNA vaccine pVKD1.0-CI (Example 3) was provided by Vekko Company of Suzhou Industrial Park.
  • mice Twenty female 6-8 week-old female BAL B / c mice were purchased from the Animal Experiment Center of Suzhou University and kept in the SPF animal room of the Animal Experiment Center of Suzhou University. The experimental animal grouping and vaccination plan are shown in Table 23. All DNA vaccines were injected into the calf anterior tibial muscle, 100 ⁇ g / head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously on the back, 10 ⁇ g / head. Two weeks after the last immunization was completed, mice were sacrificed, serum and spleen cells were collected, and subjected to an enzyme-linked immunospot assay (ELISPOT) and an enzyme-linked immunoassay (ELISA), respectively.
  • ELISPOT enzyme-linked immunospot assay
  • ELISA enzyme-linked immunoassay
  • the mouse IFN- ⁇ ELISPOT kit was purchased from BD Company (Cat. No .: 551083).
  • the stimulating peptide was NY-ESO-1 41 # peptide (WITQCFLPVFLAQPP), which was synthesized by Shanghai Ke Peptide Biotechnology Co., Ltd.
  • the final stimulating concentration was 10 ⁇ g / mL.
  • the positive stimuli phorbol-12-myristate-13-acetate (PMA) and inomysin were purchased from Sigma Company, USA.
  • the method of ELISA is well known to those skilled in the art and is briefly described below.
  • the 96-well microtiter plate was purchased from Jianghai Glass Instrument General Factory. Both the restructured LMN and NY-ESO-1 were provided by Suzhou Industrial Park Vickers Biotechnology Co., Ltd.
  • the protein was coated with NaHCO 3 buffer (pH 9.6), overnight at 4 ° C, and the coating concentration was 10 ⁇ g / mL.
  • 3,3,5,5-tetramethylbenzidine (3,3,5,5-tetraamthyl benzidine, TMB) substrate was developed, 37 ° C, 15 minutes, and terminated with 2M dilute sulfuric acid using a microplate reader (USA Thermo) read the absorbance (A) value at a wavelength of 450 nm.
  • a positive value greater than a negative control A value of 2.1 was determined, and the reciprocal of the highest dilution in the positive value was defined as the serum antibody titer. When the titer is less than the initial dilution of 1: 100, the titer is defined as 50.
  • the results of the cellular immune response test are shown in FIG. 26.
  • the primary immune-free pVKD1.0-CI-LMNB DNA vaccine, the LMNB-C5 protein boost (that is, group C in Example 11) and the LMNB-C10 protein boost (that is, group D in Example 11) have the best immune effect.
  • 5 Th epitopes (group C) and 10 Th epitopes (group D) containing CMV virus can significantly increase the cellular immune response level of weak immunogens.
  • DNA vaccine vector pVKD1.0 was provided by Suzhou Industrial Park Vickers.
  • the DNA vaccine pVKD1.0-NP expression derived from strain A / Shanghai / 02/2013 (H7N9)
  • influenza antigen NP NCBI reference sequence: YP_009118476 .1
  • the protein vaccine VP1 entityovirus 71 type VP1 protein, see Chinese patent application 201310088364.5
  • mice Sixty female female BAL B / c mice aged 6-8 weeks were purchased from the Animal Experiment Center of Soochow University and kept in the SPF animal room of the Animal Experiment Center of Soochow University. The experimental animal grouping and vaccination plan are shown in Table 24. All DNA vaccines were injected into the calf anterior tibial muscle, 100 ⁇ g / head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously on the back, 10 ⁇ g / head.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice were subcutaneously inoculated with a 4T1-hNY-ESO-1 stably transfected cell line (provided by Suzhou Industrial Park Vekida Biotechnology Co., Ltd.) at a dose of 1 ⁇ 10 5 cells / head Observe and measure tumor growth continuously after inoculation.
  • mice in each group The tumor growth of immunized mice in each group is shown in FIG. 27.
  • the mice in the control group all showed tumors on the 14th day after challenge (ie, after tumor inoculation), and grew rapidly.
  • the tumor growth of mice in each immunization group lags behind that of the control group.
  • the LMNB-I13 booster immune group group D mice and LMNB-I13 and LMNB-C10 mixed booster immune group (group E) mice have the slowest tumor growth, so These two groups of vaccines work best.
  • mice in the control group A were 14 days.
  • the tumor-free survival of mice immunized with each vaccine was significantly higher than that of the control group. It is shown that all vaccines can improve tumor-free survival of mice after immunization.
  • group D with the epitope fusion peptide of I13, and group E and F with the epitope fusion peptide of I13 and C10 had the best effect, and the tumor-free survival time of mice was doubled at most.
  • the vaccine group with the I13 epitope fusion peptide significantly improved tumor-free survival, which increased tumor-free survival by about 40%. It shows that 13 Th epitopes or 10 CMV Th epitopes loaded with influenza virus can greatly improve the tumor protection effect of tumor vaccines.
  • mice in the control group A were 35 days.
  • the overall survival of the mice immunized with each vaccine was significantly higher than that of the control group. It is shown that all vaccines can improve the survival of mice after immunization.
  • the D group with the I13 epitope fusion peptide, the E group and the F group with the I13 and C10 epitope fusion peptide had the best results, and the overall survival increased by 83%.
  • the vaccine groups with the I13 epitope fusion peptide significantly increased tumor-free survival, with a maximum increase of 28%. It shows that 13 Th epitopes or 10 CMV Th epitopes loaded with influenza virus can greatly improve the tumor protection effect of tumor vaccines.
  • Example 9 Thirty 6-8 week-old female BAL B / c mice were purchased from the Animal Experiment Center of Soochow University and kept in the SPF animal room of Animal Experiment Centre of Soochow University. The experimental animal grouping and vaccination plan are shown in Table 25. All DNA vaccines were injected into the calf anterior tibial muscle, 100 ⁇ g / head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously on the back, 10 ⁇ g / head.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice in the control group (group A) all showed tumors on the 14th day after challenge (ie, after tumor inoculation), and grew rapidly.
  • the tumor growth of the LMNB-I13 protein vaccine treatment group (group C) mice was the slowest.
  • the tumor size of the LMNB-I13 protein vaccine treatment group was significantly smaller than that of the control group (group A) on the 22nd day after the mice were challenged, and there was a significant difference in tumor size between the two groups until the 30th day.
  • tumor growth in mice in group C began to accelerate, which may be related to the cessation of vaccination of the LMNB-I13 protein vaccine.
  • the results indicate that the LMNB-I13 protein vaccine can inhibit tumor growth in mice.
  • Example 9 Thirty 6-8 week-old female BAL B / c mice were purchased from the Animal Experiment Center of Soochow University and kept in the SPF animal room of Animal Experiment Centre of Soochow University. The experimental animal grouping and vaccination plan are shown in Table 26. All DNA vaccines were injected into the calf anterior tibial muscle, 100 ⁇ g / head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously on the back, 10 ⁇ g / head.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice in the untreated control group failed to inoculate the tumor after the tumor challenge (that is, after the tumor inoculation), they were not included in the analysis and compared.
  • Mice of the parallel vaccine control group (group B) and the LMNB-I13 treatment group (group C) were presented. Compared with B, the tumor growth of mice in group C was slower, and the tumor size of the LMNB-I13 protein vaccine treatment group was significantly smaller than that of the parallel vaccine control group (group B) until the 22nd day after the mice challenged. At 30 days, there were significant differences in tumor size between the two groups.
  • mice in group C began to accelerate at a later stage, which may be related to the cessation of LMNB-I13 protein vaccine.
  • the results indicate that the LMNB-I13 protein vaccine can inhibit tumor growth in mice.
  • the I13 amino acid sequence (SEQ ID NO: 60) contains eight influenza virus Th epitopes including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and NP221 in Table 4, and Five epitopes of influenza virus including HA434, HA440, NP324, M1-127 and M1-210.
  • the eukaryotic codon-optimized sequence (SEQ ID NO: 61) encoding the amino acid sequence of I13 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and inserted into the shuttle vector pSC65 (addgene, article number: 30327) by molecular biology methods well known in the art. , Constructed into a shuttle vector pSC65-I13 containing 13 influenza epitopes.
  • a vector pVKD1.0-hLMN-CTB expressing the triple fused tumor antigens LAGE-1, MAGE-A3, NY-ESO-1 and the cholera toxin B subunit was digested and recovered by Sal I and Kpn I and inserted into the aforementioned shuttle vector pSC65-I13 to construct a shuttle vector pSC65-LMNB-I13 (the plasmid map is shown in Figure 32). After being identified by sequencing, it is stored in the library. Among them, the amino acid sequence of LMNB-I13 is shown in SEQ ID NO: 62, and its eukaryotic codon optimized sequence is shown in SEQ ID NO: 63. The restriction enzymes Sal I and Kpn I were used to identify the vector pSC65-LMNB-I13 (the digestion system is shown in Table 27). The digestion verification map is shown in Figure 33.
  • Enzyme digestion system volume Plasmid pSC65-LMNB-I13 3 ⁇ L, about 1 ⁇ g Sal I (Treasure Bio, Cat. No. 1080A) 1 ⁇ L Not I (Bao Biology, article number 1166A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH2O Fill up to 10 ⁇ L
  • the recombinant vaccinia virus vector was obtained in 143B cells, and the specific method was as follows. On the first day, spread 143B cells on a 6-well cell culture plate (JET, TCP-010-006) (supplied by Suzhou Industrial Park Vickers Biotechnology Co., Ltd.), 1 ⁇ 10 6 / well, in a 37 ° C carbon dioxide cell incubator Incubate overnight. The next day, a wild vaccinia virus strain (provided by Beijing Biological Products) was added at 0.05 MOI (that is, 5 ⁇ 10 4 PFU (plaque forming unit) / well), and then incubated in a 37 ° C carbon dioxide cell incubator for two Hours during which the shuttle vector / transfection reagent complex is prepared.
  • JET 6-well cell culture plate
  • PFU palque forming unit
  • the shuttle vector was pSC65-LMNB-I13 obtained in Example 16, and the transfection reagent was Turbofect (Thermo Fisher Scientific, R0531).
  • the transfection dose and compound method refer to the transfection reagent manual.
  • the 143B cell supernatant was changed to 2 mL / well of DMEM maintenance medium containing 2% fetal bovine serum (FBS), and then a shuttle vector / transfection reagent complex was added. 48 hours after transfection, the supernatant was removed, and the cells were collected and resuspended in 0.5 mL of maintenance medium.
  • the recombinant cell lysate was inserted into new 143B cells (containing 50 ⁇ g / mL BrdU) Incubate at 37 ° C for 1 to 2 days. Observe the cell lesions during the period, and when the appropriate number of virus plaques appear (less than 20 plaques / well), perform single-spot purification.
  • the 2% low melting point agarose is heated in a microwave oven (medium to high heat for about 2 minutes) to boiling, transferred to a 45 ° C water bath to cool down and prevent it from solidifying.
  • the recombinant vaccinia virus vector rvv-LMNB-I13 constructed in Example 17 and the wild vaccinia virus strain were separately amplified on Vero cells (provided by Suzhou Industrial Park Vekida Biotechnology Co., Ltd.).
  • the amplification method is as follows.
  • Vero monolayer cells (1 ⁇ 10 7 cells / dish) were prepared for a total of 10 dishes.
  • the cells were scraped off and collected, centrifuged at 1800 g for 5 minutes, and the supernatant was removed.
  • Resuspension was performed with 5 mL of maintenance medium, and sonication was performed on ice using an ultrasonic cell powder machine.
  • the sonication conditions were: 50 watts, 5 seconds ultrasound / 5 intervals, for a total of 15 minutes.
  • the prepared vaccinia virus was titrated for infection titer on Vero cells.
  • the specific method is as follows.
  • Table 28 shows the titer results of vaccinia virus vectors.
  • Vaccinia virus Titer PFU / mL
  • mice in the control group all showed tumors on the 15th day after tumor inoculation and grew rapidly. On the 30th day after tumor challenge, the average tumor size in the treatment group was significantly smaller than that in the control group.
  • the results indicate that vaccinia virus vector vaccine rvv-LMNB-I13 can inhibit tumor growth with MAGE-A3 expression.
  • the survival curve results of mouse survival status are shown in Fig. 35.
  • the overall survival (median OS of 36 days) of the mice treated with vaccinia virus vector vaccine rvv-LMNB-I13 was better than that of control mice (median OS of 33 days). , P ⁇ 0.05).
  • the results indicate that vaccinia virus vector vaccine rvv-LMNB-I13 can improve the survival of mice with MAGE-A3 expressing tumors.
  • mice in each group The tumor growth of immunized mice in each group is shown in FIG. 36. On the 26th day after tumor challenge, tumors in the control group grew rapidly and were significantly larger than those in the treatment group, and continued until the 50th day. The results indicate that vaccinia virus vector vaccine rvv-LMNB-I13 can inhibit tumor growth with LAGE-1 expression.
  • FIG. 37 The overall survival (median OS of 75 days) of the mice treated with vaccinia virus vector vaccine rvv-LMNB-I13 was better than that of control mice (median OS). 51 days, p ⁇ 0.05). The results indicate that vaccinia virus vector vaccine rvv-LMNB-I13 can improve the survival of mice with LAGE-1 expressing tumors.
  • the protein vaccine has a significant effect in preventing tumors (see Figures 27, 28, and 29). A certain effect can be maintained in tumor treatment, but it is not particularly obvious and cannot be sustained (see Figures 30 and 31).
  • the recombinant vaccinia virus vector has achieved particularly significant effects in the treatment of tumors. Not only has it continued to suppress tumor growth, it has also significantly improved overall survival (see Figures 34-37), with significant effects.

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Abstract

本发明提供了一种重组病毒载体、包含其的免疫组合物及其用途。所述重组病毒载体包含编码CD4辅助性T细胞表位融合肽的多核苷酸，所述表位融合肽包含巨细胞病毒表位和/或流感病毒表位。本发明提供的表位融合肽、重组病毒载体能提高目的免疫原的细胞免疫应答水平，尤其能提高弱免疫原的细胞免疫应答水平；可克服免疫系统对抗原的免疫耐受性，尤其能克服肿瘤抗原或感染相关抗原的免疫耐受性。本发明的产品适用于增强疫苗的效力。

Description

一种重组病毒载体、包含其的免疫组合物以及用途 技术领域
本发明属于分子生物学和免疫学领域。具体地，本发明涉及一种包含编码CD4辅助性T细胞表位融合肽的多核苷酸的重组病毒载体、包含其的免疫组合物以及用途。
背景技术
T辅助细胞(Th细胞)是一种在免疫系统中起重要作用的T细胞，特别是在适应性免疫系统中。它们通过释放T细胞细胞因子来帮助其他免疫细胞的活性。这些细胞有助于抑制或调节免疫反应。它们在B细胞抗体类别的转换、细胞毒性T细胞的活化和生长以及吞噬细胞如巨噬细胞的杀菌活性最大化中是必需的。
成熟Th细胞表达蛋白CD4，称为CD4 +T细胞。通常将这样的CD4 +T细胞作为免疫系统内的辅助T细胞进行预定义的处理。例如，当抗原呈递细胞在II类MHC上表达抗原时，CD4 +细胞将通过细胞与细胞相互作用的组合(例如CD40(蛋白质)和CD40L)和细胞因子来帮助这些细胞。
可以从HIV(一种主要感染CD4 +T细胞的病毒)中看到辅助T细胞的重要性。在艾滋病毒感染的晚期阶段，功能性CD4 +T细胞的丧失导致被称为获得性免疫缺陷综合征(AIDS)的感染症状阶段。当血液或其他体液早期发现HIV病毒时，连续治疗可能会延迟发生这种情况的时间。如果发生艾滋病，治疗也可以更好地管理艾滋病的过程。还有其他罕见的疾病，如淋巴细胞减少症，导致CD4 +T细胞的缺失或功能障碍。这些疾病产生类似的症状，其中许多是致命的。
抗原表位(antigenic epitope)，简称“表位”(epitope)，也称为“抗原决定簇”(antigenic determinant)，是指抗原表面上决定抗原特异性的化学基团。抗原表位可被免疫系统(尤其是抗体、B细胞或者T细胞)所识别。抗体中能识别抗原表位的区域叫做“互补位”或“抗体决定簇”。尽管通常抗原表位是指外来蛋白质等物质的其中一部分，但只要能被自身免疫系统所识别的表位，也被归为抗原表位。
蛋白质抗原的表位根据它们的结构以及与互补位的交互作用，被分为构象表位和线性表位这两种类型。其中构象表位由抗原氨基酸序列中的不连续部分组成，因此互补位和抗原表位的交互作用是基于表面的三维特征和形状，或者是抗原的三级结构。大部分的抗原表位都属于构象表位。与此相反，线性表位是由一段连续的抗原氨基酸序列构成，与抗原的交互作 用的基础是其一级结构。
T细胞表位(T cell epitope)主要是由8-17氨基酸组成的短肽构成，出现在抗原呈递细胞(antigen-presenting cell,APC)上，这种抗原表位将会和主要组织相容性复合体(MHC)相结合形成复合体，并与相应的T细胞表位受体结合，从而活化T细胞，产生相应细胞免疫应答(Shimonkevitz et al.,1984；Babbitt et al.,1985；Buus et al.,1986；Townsend and Bodmer,1989)。与表位结合的MHC主要有两类分子，其中，I类主要组织相容性复合体所呈现的T细胞抗原表位通常是由8至11个氨基酸长度的多肽，而II类主要组织相容性复合体所呈现的T细胞抗原表位相对更长，由13-17个氨基酸组成。
辅助T细胞表位(Th epitope，即Th表位)是指T细胞表位中的，与MHC分子结合形成的复合体能被CD4辅助性T细胞受体识别的一类T细胞表位。Th表位主要与存在于由主要组织相容性复合体(MHC)的II类基因编码的抗原呈递细胞(antigen-presenting cell,APC)表面上的分子结合。然后，II类分子和肽表位的复合物被T辅助淋巴细胞表面上的特异性T细胞受体(TCR)识别。以这种方式，在MHC分子的环境中呈现抗原表位的T细胞可以被激活，并为B淋巴细胞分化提供必要的信号。传统上，肽免疫原的辅助T细胞表位的来源是肽共价偶联的载体蛋白，但是该偶联过程可引入其他问题，例如在偶联过程中抗原决定簇的修饰和以针对肽的抗体为代价的针对载体的抗体的诱导(Schutze,M.P.,Leclerc,C.Jolivet,M.Audibert,F.Chedid,L.Carrier-induced epitopic suppression,a major issue for future synthetic vaccines.J Immunol.1985,135,2319-2322；DiJohn,D.,Torrese,J.R.Murillo,J.Herrington,D.A.et al.Effect of priming with carrier on response to conjugate vaccine.The Lancet.1989,2,1415-1416)。此外，在制备中使用无关蛋白引入质量控制问题。合适的载体蛋白的选择在设计肽疫苗时非常重要，其选择受到其大规模生产的毒性和可行性等因素的限制。这种方法还有其他限制，包括可偶联的肽负载的大小和可以安全施用的载体的剂量(Audibert,F.a.C.,L.1984.Modern approaches to vaccines.Molecular and chemical basis of virus virulence and immunogenicity.,Cold Spring Harbor Laboratory,New York.)。虽然载体分子允许诱导强的免疫应答，但它们也与不利影响相关，例如抑制抗肽抗体应答(Herzenberg,L.A.and Tokuhisa,T.1980.Carrier-priming leads to hapten-specific suppression.Nature 285:664；Schutze,M.P.,Leclerc,C.,Jolivet,M.,Audibert,F.,and Chedid,L.1985.Carrier-induced epitopic suppression,a major issue for future synthetic vaccines.J Immunol 135:2319； Etlinger,H.M.,Felix,A.M.,Gillessen,D.,Heimer,E.P.,Just,M.,Pink,J.R.,Sinigaglia,F.,Sturchler,D.,Takacs,B.,Trzeciak,A.,and et al.,1988.Assessment in humans of a synthetic peptide-based vaccine against the sporozoite stage of the human malaria parasite,Plasmodium falciparum.J Immunol 140:626)。
一般来说，免疫原必须包含辅助T细胞表位，除了将被表面Ig识别的表位或存在于细胞毒性T细胞上的受体之外。应该意识到，这些类型的表位可能是非常不同的。对于B细胞表位，构象是重要的，因为B细胞受体直接与天然免疫原结合。相比之下，由T细胞识别的表位不依赖于表位的构象完整性，并且由针对CTL的约9个氨基酸的短序列和针对辅助T细胞的稍长的序列(长度限制较小)组成。这些表位的唯一要求是它们可以分别容纳在I类或II类分子的结合裂口中，并且然后复合物能够与T细胞受体结合。II类分子的结合位点在两端是开放的，允许与报道的短至8个氨基酸残基(Fahrer,A.M.,Geysen,H.M.,White,D.O.,Jackson,D.C.and Brown,L.E.Analysis of the requirements for class II-restricted T-cell recognition of a single determinant reveals considerable diversity in the T-cell response and degeneracy of peptide binding to HEd J.Immunol.1995.155:2849-2857)的表位结合的肽的长度变化更大(Brown,J.H.,T.S.Jardetzky,J.C.Gorga,L.J.Stern,R.G.Urban,J.L.Strominger and D.C.Wiley.1993.Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1.Nature 364:33)。
Th表位能刺激并活化辅助T细胞，相应地，促进CD8T细胞和B细胞活化，最终提高免疫应答。实质上，Th表位除了能活化针对自身的免疫应答外，还能有效帮助与之关联的其他抗原或表位的免疫应答。因此，可以将异源性的强Th表位与目的免疫原融合，据此可以提高目的免疫原的免疫原性。一种称之为“PADRE(pan HLA DR-binding Epitope)”的人造强Th表位已用于多个疫苗的融合构造，以提高相关免疫原的免疫应答水平(del Guercio et al.,Vaccine,1997,15:441.；Franke,E.D.et al.,Vaccine,1999,17:1201；Jeff Alexander et al.,J Immunol,2000,164(3)1625-1633；Jeff Alexander et al.,Vaccine,2004,22:2362.；La Rosa,Corinna et al.,The Journal of infectious diseases,2012,205:1294-304)。此外，源于破伤风毒素(Tetanus toxin)的强Th表位P2也常用于与目的免疫原偶联，以提高免疫原性(Panina-Bordignon P et al.,Eur J Immunol,1989,19:2237-42；La Rosa,Corinna et al.,The Journal of infectious diseases,2012,205:1294-304)。
但一般而言，用于提高免疫原性的Th表位通常为异源性的，换言之， 疫苗受试者体内不会存在针对该Th表位自身的高水平的免疫应答。因此，使用上述的强Th表位接种疫苗受试者时，很可能的情况是疫苗受试者的免疫系统是初次接触此类Th表位，受体免疫系统活化针对此类Th表位和目的免疫原的表位基本上是同步的，针对此类Th表位的T细胞的生成时间和数量与目的免疫原相似，这样的话，对于帮助目的免疫原的效果会因此受限。特别是对于弱免疫原性的肿瘤抗原，此类Th表位的辅助作用更难发挥。事实上，直接采用强Th表位虽然能活化肿瘤抗原，但其激发的细胞免疫应答水平仍较低，不能满足肿瘤疫苗的需要(Ghaffari-Nazari H et al.,PLoS ONE,2015,10(11):e0142563)。
因此，需要新的Th表位策略来提高目的免疫原免疫原性，特别是一些弱的免疫原，例如肿瘤抗原。
发明内容
本发明的目的是提供一种CD4辅助性T细胞表位融合肽，通过该表位融合肽，目的免疫原的免疫原性得以提高。
进一步地，本发明利用来源于巨细胞病毒(Cytomegalovirus，CMV)和流感病毒(Influvirus,Flu)的强Th表位获得表位融合肽来提高目的免疫原的免疫原性。
为了本发明的目的以下定义下列术语。
“表位融合肽”是指将若干表位连接在一起所形成的一个肽。
“目的免疫原”是指为实现某种免疫应答，所采用的免疫原，包括抗原等具有免疫学活性的物质，优选为蛋白。
本发明的另一目的是提供一种所述表位融合肽和所述目的免疫原的融合蛋白。
为实现上述目的，本发明提供一种CD4辅助性T细胞表位融合肽，其包含巨细胞病毒表位和/或流感病毒表位。本发明还提供一种重组病毒载体，所述重组病毒载体包含编码CD4辅助性T细胞表位融合肽的多核苷酸，所述CD4辅助性T细胞表位融合肽包含巨细胞病毒表位和/或流感病毒表位。
在本发明的一个实施方案中，所述表位融合肽包含选自SEQ ID NO:1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO:11-23所示的流感病毒表位中的一种或多种。
在本发明的一个实施方案中，所述表位融合肽由选自SEQ ID NO：1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO：11-23所示的流感病毒表位中的一种或多种组成。优选地，所述表位融合肽由5个 或10个巨细胞病毒表位和/或由8个或13个流感病毒表位组成，例如SEQ ID NO:34或44所示的表位融合肽。最优选地，所述表位融合肽由13个流感病毒表位组成，例如SEQ ID NO:48或60所示的表位融合肽。
优选地，所述表位融合肽诱导体液或细胞免疫应答。
优选地，重组病毒是痘苗病毒，优选为复制型痘苗病毒载体，例如痘苗病毒天坛株，例如752-1株，或者为非复制型痘苗病毒载体，例如痘苗病毒减毒疫苗安卡拉株(Modified Vaccinia Ankara，MVA)。
本发明还提供了所述表位融合肽和目的免疫原的融合蛋白。
本发明还提供了编码所述表位融合肽和/或所述融合蛋白的多核苷酸。
在本发明的一个实施方案中，所述目的免疫原为任意一种或多种免疫原。优选的，所述目的免疫原为肽、抗原、半抗原、碳水化合物、蛋白质、核酸、过敏原、病毒或病毒的一部分、细菌、寄生虫或其它完整的微生物。
在本发明的一个实施方案中，所述抗原为肿瘤抗原或感染相关抗原。
在本发明的一个实施方案中，所述肿瘤抗原选自肺癌抗原、睾丸癌抗原或黑色素瘤抗原、肝癌抗原、乳腺癌抗原或前列腺癌抗原中的一种或多种。
在本发明的一个实施方案中，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种。优选地，LAGE抗原为LAGE-1，MAGE抗原为MAGE-A3。进一步优选地，所述肿瘤抗原包含LAGE-1、MAGE-A3和NY-ESO-1。优选地，所述LAGE-1的氨基酸序列如SEQ ID NO:24所示，所述MAGE-A3的氨基酸序列如SEQ ID NO:25所示，所述NY-ESO-1的氨基酸序列如SEQ ID NO:26所示；还进一步优选地，所述肿瘤抗原包含LAGE-1、MAGE-A3和NY-ESO-1。
在本发明的一个实施方案中，所述感染相关抗原选自HIV抗原、流感病毒抗原或HBV抗原中的一种或多种。
优选地，所述融合蛋白如SEQ ID NO:55-58、62中之一所示。
优选地，所述融合蛋白的编码基因如SEQ ID NO:63所示。
本发明的另一目的是提供一种免疫组合物，所述免疫组合物包含治疗有效量的根据本发明的表位融合肽、融合蛋白、多核苷酸和/或重组病毒载体，以及药学上可接受的载体。优选地，该免疫组合物为疫苗。
本发明的另一目的是提供一种药盒，所述药盒包含根据本发明的表位融合肽、融合蛋白、多核苷酸、重组病毒载体和/或免疫组合物，以及其使用说明。
本发明还提供了根据本发明的表位融合肽、融合蛋白、多核苷酸、重组病毒载体和/或免疫组合物在制备提高目的免疫原的免疫原性的药物或 疫苗中用途。
本发明还提供了一种使用根据本发明的表位融合肽提高目的免疫原的免疫原性的方法，包括将疫苗受试者或人群中已有较强免疫应答的CD4辅助性T细胞表位与目的免疫原融合形成的融合蛋白。其方法具体为：
(1)选择一个或多个CD4辅助性T细胞的表位，所述表位与MHC分子结合形成的复合体能被CD4辅助性T细胞受体识别，并且在疫苗接种前，疫苗受试者体内已经存在针对所述表位中至少一个表位产生了T细胞免疫应答；
(2)将上述表位融合以制成表位融合肽，将所述表位融合肽与目的免疫原融合以制成融合蛋白，表达融合蛋白并制成疫苗，其表达载体形式可以是DNA疫苗载体形式，可以是蛋白疫苗载体形式，还可以是病毒疫苗载体形式；
(3)将上述疫苗接种给疫苗受试者，接种时可以选择适当的佐剂，例如不完全弗氏佐剂，完全弗氏佐剂，氢氧化铝佐剂等。
进一步地，所述使用方法的步骤(1)还包括检查疫苗受试者MHC表型的步骤。优选地，检查疫苗受试者MHC表型包括检查疫苗受试者的MHC II类基因亚型型别。
本发明还提供了一种治疗或预防有需要的受试者的病症的方法，其包括给予治疗有效量的本发明的表位融合肽、免疫组合物、多核苷酸和/或重组病毒载体。优选地，所述病症选自恶性肿瘤、细菌和病毒性感染中的一种或多种。优选地，所述恶性肿瘤为乳腺癌或结肠癌。优选地，所述感染是慢性感染。优选地，该方法中，初免使用DNA疫苗载体，加强免疫使用蛋白疫苗载体，更优选地，初免使用pVKD1.0-CI-LMNB DNA疫苗，加强免疫使用LMNB-I13蛋白。
本发明提供的表位融合肽能大幅度提高目的免疫原，尤其是弱免疫原的细胞免疫应答水平，是克服免疫系统对抗原免疫耐受性，尤其是对肿瘤抗原或感染相关抗原免疫耐受性的有效手段，适用于高效增强疫苗的效力。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1和图2分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原编码序列的DNA疫苗载体pVKD1.0-hLMN的质粒图谱和双酶切鉴定图。
图3和图4分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的DNA疫苗载体pVKD1.0-hLMN-CTB的质粒 图谱和双酶切鉴定图。
图5和图6分别是带有源于CMV和流感病毒CD4表位编码序列的DNA疫苗载体pVKD1.0-CI的质粒图谱和双酶切鉴定图。
图7和图8分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位，和源于CMV和流感病毒CD4表位编码序列的DNA疫苗载体pVKD1.0-CI-LMNB的质粒图谱和双酶切鉴定图。
图9和图10分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原编码序列的原核载体pET-30a(+)-LMN的质粒图谱和双酶切鉴定图。
图11和图12分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-LMN-CTB的质粒图谱和双酶切鉴定图。
图13和图14分别是带有源于CMV表位编码序列的原核载体pET-30a(+)-CMV Th的质粒图谱和双酶切鉴定图。
图15和图16分别是带有源于CMV表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-CMV10-LMNB的质粒图谱和双酶切鉴定图。
图17和图18分别是带有源于流感病毒表位编码序列的原核载体pET-30a(+)-CMV Th的质粒图谱和双酶切鉴定图。
图19和图20分别是带有源于流感病毒表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-Influ8-LMNB的质粒图谱和双酶切鉴定图。
图21和图22分别是带有源于流感病毒表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-Influ13-LMNB的质粒图谱和双酶切鉴定图。
图23为动物免疫实验中的细胞免疫应答检测结果。
图24和图25分别是带有源于CMV表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-CMV5-LMNB的质粒图谱和双酶切鉴定图。
图26为实施例12中的动物免疫实验中的细胞免疫应答检测结果。
图27为实施例13中的小鼠肿瘤生长情况。
图28和图29分别是实施例13中的小鼠无瘤存活状况和总体存活状况。
图30为4T1-hNY-ESO-1小鼠肿瘤模型中，各治疗小组小鼠肿瘤生长情况。
图31为CT26-hLAGE-1小鼠肿瘤模型中，各治疗小组小鼠肿瘤生长 情况。
图32和图33分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原编码序列和13个流感表位编码序列(I13)的穿梭载体载体pSC65-LMNB-I13的质粒图谱和双酶切鉴定图。
图34为实施例19中的CT26-MAGE-A3小鼠肿瘤模型中，各组小鼠肿瘤生长情况。
图35为实施例19中的CT26-MAGE-A3小鼠肿瘤模型中，各组小鼠总体存活状况。
图36为实施例20中的CT26-LAGE-1小鼠肿瘤模型中，各组小鼠肿瘤生长情况
图37为实施例20中的CT26-LAGE-1小鼠肿瘤模型中，各组小鼠总体存活状况。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
实施例1 DNA疫苗pVKD1.0-hLMN构建
LAGE-1、MAGE-A3和NY-ESO-1氨基酸序列分别如SEQ ID NO:24-26所示。通过在线密码子优化软件(http://www.jcat.de/)将上述抗原氨基酸序列优化成哺乳动物密码子使用偏好的核苷酸序列，分别如SEQ ID NO:27-29所示。经上海捷瑞生物技术有限公司合成后，通过本领域熟知的方法克隆至DNA疫苗载体pVKD1.0(苏州工业园区唯可达生物科技有限公司提供)上的多克隆位点Sal I与BamH I之间，构建成可表达融合蛋白抗原的DNA疫苗载体pVKD1.0-hLMN(质粒图谱如图1)，经测序鉴定正确后入库。用限制内切酶Sal I与BamH I鉴定载体pVKD1.0-hLMN(酶切体系如表1)，其酶切验证图谱如图2所示。
表1：质粒pVKD1.0-hLMN的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-hLMN 3μL，约1μg
Sal I(宝生物，货号1080A) 1μL
BamH I(宝生物，货号1010A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例2 DNA疫苗pVKD1.0-hLMN-CTB构建
霍乱毒素B亚单位(Cholera toxin subunit B，简称CTB)的氨基酸序列(SEQ ID NO:30)的哺乳动物密码子优化序列(SEQ ID NO:31)及其真核表达载体pVKD1.0-CTB由苏州工业园区唯可达生物科技有限公司提供。以pVKD1.0-CTB为模板，设计引物(表2)，通过PCR扩增CTB基因片段，然后凝胶回收相应片段，通过同源重组法将CTB片段插入线性化载体pVKD1.0-hLMN相应位置上，构建成DNA疫苗载体pVKD1.0-hLMN-CTB(质粒图谱如图3)，经测序鉴定正确后入库。用限制内切酶Sal I与BamH I鉴定载体pVKD1.0-hLMN-CTB(酶切体系如表3)，其酶切验证图谱如图4所示。
表2：实施例2中的引物
Figure PCTCN2019090877-appb-000001
表3：质粒pVKD1.0-hLMN-CTB的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-hLMN-CTB 3μL，约1μg
Sal I(宝生物，货号1080A) 1μL
BamH I(宝生物，货号1010A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例3 DNA疫苗pVKD1.0-CI-LMNB构建
在免疫表位数据库(IEDB，http://www.iedb.org)上获取来源于巨细胞病毒(Cytomegalovirus，CMV)和流感病毒(Influvirus,Flu)的强Th表位(见表4)，其中，CMV的强Th表位包括pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104；流感病毒的强Th表位包括HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221、HA434、HA440、NP324、M1-127和M1-210。表4中选择的表位覆盖人群中MHC II类分子多数亚型，而且也覆盖小鼠MHC II亚型分子。然后将所选的表位pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221串联在一起，形成一段CMV病毒和流感病毒的表位融合肽，其氨基酸序列如SEQ ID NO:34 所示，该表位融合肽经哺乳动物密码子优化后，核酸序列如SEQ ID NO:35所示。送苏州泓迅生物科技有限公司合成该核酸序列，然后通过本领域熟知的分子生物学方法将该核酸序列插入至DNA疫苗载体pVKD1.0(苏州工业园区唯可达生物科技有限公司)上，形成载体pVKD1.0-CI(质粒图谱如图5)，经测序鉴定正确后入库。用限制内切酶Pst I与Bgl II鉴定载体pVKD1.0-CI(酶切体系如表5)，其酶切验证谱如图6所示。
表4：实施例3中的Th表位
表位名称 来源 氨基酸序列
pp65-11 CMV LLQTGIHVRVSQPSL(SEQ ID NO:1)
pp65-71 CMV IIKPGKISHIMLDVA(SEQ ID NO:2)
pp65-92 CMV EHPTFTSQYRIQGKL(SEQ ID NO:3)
pp65-123 CMV AGILARNLVPMVATV(SEQ ID NO:4)
pp65-128 CMV KYQEFFWDANDIYRI(SEQ ID NO:5)
pp65-57 CMV KVYLESFCEDVPSGK(SEQ ID NO:6)
pp65-62 CMV TLGSDVEEDLTMTRN(SEQ ID NO:7)
pp65-30 CMV PLKMLNIPSINVHHY(SEQ ID NO:8)
pp65-112 CMV ACTSGVMTRGRLKAE(SEQ ID NO:9)
pp65-104 CMV TERKTPRVTGGGAMA(SEQ ID NO:10)
HA203 Influ NQRALYHTENAYVSVVS(SEQ ID NO:11)
NP438 Influ SDMRAEIIKMMESARPE(SEQ ID NO:12)
NS1-84 Influ ALASRYLTDMTIEEMSR(SEQ ID NO:13)
M1-181 Influ LASTTAKAMEQMAGSSE(SEQ ID NO:14)
HA375 Influ SGYAADQKSTQNAINGITNKVN(SEQ ID NO:15)
NP24 Influ EIRASVGKMIDGIGRFYI(SEQ ID NO:16)
NP95 Influ PIYRRVDGKWMRELVLY(SEQ ID NO:17)
NP221 Influ RMCNILKGKFQTAAQRAM(SEQ ID NO:18)
HA434 Influ IWTYNAELLVLLENERT(SEQ ID NO:19)
HA440 Influ ELLVLLENERTLDFHDS(SEQ ID NO:20)
NP324 Influ HKSQLVWMACNSAAFED(SEQ ID NO:21)
M1-127 Influ CMGLIYNRMGAVTTESA(SEQ ID NO:22)
M1-210 Influ RQMVQAMRAIGTHPSSSTGLKND(SEQ ID NO:23)
表5：质粒pVKD1.0-CI的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-CI 3μL，约1μg
Pst I(宝生物，货号1073A) 1μL
Bgl II(宝生物，货号1021A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
最后以实施例2中的载体pVKD1.0-hLMN-CTB为模板，设计引物(表6)，通过PCR扩增hLMN-CTB目的基因片段，然后通过本领域熟知的分子生物学方法将该基因片段插入该pVKD1.0-CI载体上Not I和Bam HI酶切位点之间，构建成DNA疫苗载体pVKD1.0-CI-LMNB(质粒图谱如图7)，经测序鉴定正确后入库。用限制内切酶Bam HI与EcoR V鉴定载体pVKD1.0-CI-LMNB(酶切体系如表7)，其酶切验证图谱如图8所示。
表6：实施例3中的引物
Figure PCTCN2019090877-appb-000002
表7：质粒pVKD1.0-CI-LMNB的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-CI-LMNB 3μL，约1μg
Bam HI(宝生物，货号1010A) 1μL
EcoR V(宝生物，货号1042A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例4 LMN原核表达载体构建
LAGE-1、MAGE-A3和NY-ESO-1氨基酸序列分别如SEQ ID NO:24-26所示。通过在线密码子优化软件(http://www.jcat.de/)将所述抗原氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列，其核苷酸序列分别如SEQ ID NO:38-40所示。经苏州泓迅生物科技有限公司合成后，通过本领域熟知 的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成原核表达构建体pET-30a(+)-LMN(质粒图谱如图9)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-LMN(酶切体系如表8)，其酶切验证图谱如图10所示。
表8：质粒pET-30a(+)-LMN的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-LMN 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例5 LMN-CTB原核表达载体构建
霍乱毒素B亚单位CTB氨基酸序列(SEQ ID NO:30)及其原核密码子优化核酸序列(SEQ ID NO:41)均由苏州工业园区唯可达生物科技有限公司提供。设计引物(表9)，以pET-30a(+)-CTB(苏州工业园区唯可达生物科技有限公司)为模板，用PCR方法扩增含有CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过同源重组的方式将该核酸片段插入pET-30a(+)-LMN载体上，构建成pET-30a(+)-LMN-CTB载体(质粒图谱如图11)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-LMN-CTB(酶切体系如表10)，其酶切验证图谱如图12所示。
表9：实施例5中的引物
Figure PCTCN2019090877-appb-000003
表10：实施例5中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-LMN-CTB 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例6含有LMN-CTB与CMV Th表位融合蛋白的原核表达载体构建
从表4中选取10个来源于CMV的Th表位pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104，将其串联在一起，所组成的氨基酸序列如SEQ ID NO:44所示，其中，该SEQ ID NO:44中的一段序列“EFELRRQ”是因为引入了酶切位点导致的，属于融合构建常用的技术。通过在线密码子优化软件(http://www.jcat.de/)将所述Th表位氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列(SEQ ID NO:45)，经上海捷瑞生物技术有限公司合成后，通过本领域熟知的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成可表达融合蛋白抗原的原核表达构建体pET-30a(+)-CMV Th(质粒图谱如图13)，经测序鉴定正确后入库。用限制内切酶Mlu I和Xho I鉴定载体pET-30a(+)-CMV Th(酶切体系如表11)，其酶切验证图谱如图14所示。
其中，如图13所示，CMV Th1含有5个CMV的Th表位，由pp65-11、pp65-71、pp65-92、pp65-123和pp65-128串联而成，CMV Th2含有5个CMV的Th表位，由pp65-57、pp65-62、pp65-30、pp65-112和pp65-104组成，CMV Th1和CMV Th2之间引入了EcoR I、Sac I和Sal I等3个限制酶酶切位点。
表11：质粒pET-30a(+)-CMV Th的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV Th 3μL，约1μg
Mlu I(宝生物，货号1071A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
设计引物(表12)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例6中的pET-30a(+)-CMV Th载体上的Not I和Xho I之间，构建成pET-30a(+)-CMV10-LMNB载体(质粒图谱如图15，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV10-LMNB(酶切体系如表13)，其酶切验证图谱如图16所示。如图15所示，pET-30a(+)-CMV10-LMNB含有CMV Th1和CMV Th2片段， 即该载体含有表4中所有10个CMV Th表位。这些表位是pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104。
表12：实施例6中的引物设计
引物 序列
6F(SEQ ID NO:46) GCGCGGCCGCGACGACAAGGCCATGGCT
6R(SEQ ID NO:47) GCCTCGAGGTTAGCCATAGAGATAGC
表13：pET-30a(+)-CMV10-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV10-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例7含有LMN-CTB与Influ Th表位融合蛋白的原核表达载体构建
从表4中选取13个来源于流感病毒的Th表位HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221、HA434、HA440、NP324、M1-127和M1-210，将其串联在一起，所组成的氨基酸序列如SEQ ID NO:48所示。
通过在线密码子优化软件(http://www.jcat.de/)将所述含有流感病毒Th表位的氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列(SEQ ID NO:49)，经上海捷瑞生物技术有限公司合成后，通过本领域熟知的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成可表达融合蛋白抗原的原核表达构建体pET-30a(+)-Influ Th(质粒图谱如图17)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-Influ Th(酶切体系如表14)，其酶切验证图谱如图18所示。
其中，如图17所示，Influ Th1含有8个流感病毒的Th表位，由HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221串联而成，Influ Th2含有5个流感病毒的Th表位，由HA434、HA440、NP324、M1-127和M1-210组成，Influ Th1和Influ Th2之间引入了EcoR I、Sac I和Sal I等3个限制酶酶切位点。
表14：实施例7中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ Th 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
设计引物(表15)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例7中的pET-30a(+)-Influ Th载体上的Not I和Sal I之间，构建成pET-30a(+)-Influ8-LMNB载体(含8个流感病毒Th表位，质粒图谱如图19)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-Influ8-LMNB(酶切体系如表16)，其酶切验证图谱如图20所示。
如图19所示，pET-30a(+)-Influ8-LMNB载体含有Influ Th1片段，即含有包括表4中的HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221在内的8个流感病毒Th表位。
表15：实施例7中的引物
Figure PCTCN2019090877-appb-000004
表16：质粒pET-30a(+)-Influ8-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ8-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
设计引物(表17)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法 将该核酸片段分别插入实施例6中的pET-30a(+)-Influ Th载体上的Not I和Xho I之间，构建成pET-30a(+)-Influ13-LMNB载体(含13个流感病毒Th表位，质粒图谱如图21)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV10-LMNB(酶切体系如表18)，其酶切验证图谱如图22所示。
如图21所示，pET-30a(+)-Influ13-LMNB载体含有Influ Th1和Influ Th2片段，即含有包括表4中的HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221在内的8个流感病毒Th表位和包括表4中的HA434、HA440、NP324、M1-127和M1-210在内的5个流感病毒Th表位。该载体总共包括表4中的所有13个流感病毒Th表位。
表17：实施例7中的引物设计
引物 序列
7F2(SEQ ID NO:52) GCCTCGAGGTTAGCCATAGAGATAGCA
7R2(SEQ ID NO:53) GCGCGGCCGCGACGACAAGGCCATGGCTATG
表18：实施例7中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ13-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例8融合蛋白的表达纯化
分别将实施例4中构建的原核表达载体pET-30a(+)-LMN、实施例5中构建的原核表达载体pET-30a(+)-LMN-CTB、实施例6中构建的原核表达载体pET-30a(+)-CMV5-LMNB和pET-30a(+)-CMV10-LMNB、实施例7中构建的原核表达载体pET-30a(+)-Influ8-LMNB和pET-30a(+)-Influ13-LMNB转化至BL21(DE3)感受态细胞(天根生化科技(北京)有限公司，货号CB105，转化方法参见感受态细胞说明书)，根据《pET系统手册》(TB055 8th Edition02/99，Novagen)分别制备重组蛋白LMN(其氨基酸序列如SEQ ID NO:59所示)、LMNB(其氨基酸序列如SEQ ID NO:54所示)、LMNB-C10(其氨基酸序列如SEQ ID NO:58所示)、LMNB-I8(其氨基酸序列如SEQ ID NO:55所示)和LMNB-I13(其氨基酸序列如SEQ ID NO:56所示)，所制蛋白分装后，-80℃保存。
经BCA法(碧云天生物技术研究所，货号P0009)检测，所制备的重组蛋白浓度为1mg/mL，检测方法参看检测试剂盒说明书。经凝胶法(厦门鲎试剂实验厂有限公司，货号G011000)检测，所制备的重组蛋白内毒素含量<1EU/mg，符合动物实验要求，检测方法参看鲎试剂说明书。
实施例9动物免疫实验
实施例2、3以及实施例8所制备的疫苗信息如表19。DNA疫苗载体pVKD1.0由苏州工业园区唯可达公司提供，DNA疫苗pVKD1.0-NP(表达源于毒株A/Shanghai/02/2013(H7N9))的流感抗原NP(NCBI参考序列:YP_009118476.1)由苏州工业园区唯可达公司提供，蛋白疫苗VP1(肠道病毒71型VP1蛋白，参见中国专利申请201310088364.5)由苏州工业园区唯可达公司提供。
从苏州大学动物实验中心购买16只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表20。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，处死小鼠，收集血清和脾细胞，分别进行酶联免疫斑点实验(ELISPOT)和酶联免疫实验(ELISA)。
小鼠IFN-γELISPOT试剂盒购自美国BD公司(货号：551083)，方法参见BD公司IFN-γELISPOT试剂盒说明书。刺激肽为NY-ESO-1 41#肽(WITQCFLPVFLAQPP)，为上海科肽生物科技有限公司合成，刺激终浓度为10μg/mL。阳性刺激物佛波醇-12-肉豆蔻酯-13-乙酯(phorbol-12-myristate-13-acetate,PMA)和离子霉素(inomysin)均购自美国Sigma公司。
ELISA的方法为本领域人员熟知，简述如下。96孔酶标板购自江海玻璃仪器总厂。重组LMN和NY-ESO-1均由苏州工业园区唯可达生物科技有限公司提供。用NaHCO 3缓冲液(pH 9.6)包被蛋白，4℃过夜，包被浓度为10μg/mL。用含0.1％牛血清白蛋白(bovine serum albumin,BSA)的磷酸盐缓冲液(PBS)37℃封闭30分钟，再用含0.5％吐温20的磷酸盐缓冲液(PBST)洗5遍后，室温孵育小鼠血清1小时，初始稀释度为1:100，PBST洗5遍后，1:5000孵育羊抗鼠HRP二抗(美国Santacruz公司)，37℃、30分钟，PBST洗5遍后用3,3,5,5-四甲基联苯胺(3,3,5,5-tetraamthyl benzidine,TMB)底物显色，37℃、15分钟，用2M稀硫酸终止后用酶标仪(美国Thermo公司)在450nm波长下读取吸光度(A)值。大于阴性对照A值2.1倍判断为阳性值，阳性值中的最高稀释度的倒数定义为血清抗体滴度。滴度小于起始稀释度1:100时，定义其滴度 为50。
表19：疫苗信息
Figure PCTCN2019090877-appb-000005
表20：分组与免疫规划
Figure PCTCN2019090877-appb-000006
细胞免疫应答检测结果如图23所示。其中，初免pVKD1.0-CI-LMNB DNA疫苗，LMNB-I13蛋白加强(即实施例8中的D组)免疫效果最好，显著性高于其平行对照(B组)以及LMNB-I8加强组(C组))。而且LMNB-I13蛋白加强组的细胞免疫应答水平与平行对照组(B组)相比，提高了将近3倍。表明装有流感病毒13个Th表位(D组)能大幅度提高弱免疫原的细胞免疫应答水平。
实施例10含有LMN-CTB与CMV Th表位融合蛋白的原核表达载体构建
设计引物(表21)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例6中的pET-30a(+)-CMV Th载体上的Not I和Sal I之间，构建成pET-30a(+)-CMV5-LMNB载体(质粒图谱如图24)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV5-LMNB(酶切体系如表22)，其酶切验证图谱如图25所示。如图24所示，pET-30a(+)-CMV5-LMNB含有CMV Th1片段，即该载体含有表4中前5个CMV Th表位。这些表位是pp65-11、pp65-71、pp65-92、pp65-123和pp65-128。
表21：实施例10中的引物设计
引物 序列
7F1(SEQ ID NO:50) GCGCGGCCGCGTTAGCCATAGAGATAGC
7R1(SEQ ID NO:51) GCGTCGACAAGACGACAAGGCCATGGCTATGC
表22：pET-30a(+)-CMV10-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV10-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例11融合蛋白的表达纯化
如实施例8所述，将实施例10中构建的原核表达载体pET-30a(+)-CMV5-LMNB转化至BL21(DE3)感受态细胞(天根生化科技(北京)有限公司，货号CB105，转化方法参见感受态细胞说明书)，根据《pET系统手册》(TB055 8th Edition02/99，Novagen)制备重组蛋白LMNB-C5(其氨基酸序列如SEQ ID NO:57所示)，所制蛋白分装后，-80℃保存。
经BCA法(碧云天生物技术研究所，货号P0009)检测，所制备的重组蛋白浓度为1mg/mL，检测方法参看检测试剂盒说明书。经凝胶法(厦门鲎试剂实验厂有限公司，货号G011000)检测，所制备的重组蛋白内毒素含量 <1EU/mg，符合动物实验要求，检测方法参看鲎试剂说明书。
实施例12动物免疫实验
疫苗信息如表19。DNA疫苗pVKD1.0-CI(实施例3)由苏州工业园区唯可达公司提供。
从苏州大学动物实验中心购买20只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表23。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，处死小鼠，收集血清和脾细胞，分别进行酶联免疫斑点实验(ELISPOT)和酶联免疫实验(ELISA)。
小鼠IFN-γELISPOT试剂盒购自美国BD公司(货号：551083)，方法参见BD公司IFN-γELISPOT试剂盒说明书。刺激肽为NY-ESO-1 41#肽(WITQCFLPVFLAQPP)，为上海科肽生物科技有限公司合成，刺激终浓度为10μg/mL。阳性刺激物佛波醇-12-肉豆蔻酯-13-乙酯(phorbol-12-myristate-13-acetate,PMA)和离子霉素(inomysin)均购自美国Sigma公司。
ELISA的方法为本领域人员熟知，简述如下。96孔酶标板购自江海玻璃仪器总厂。重组LMN和NY-ESO-1均由苏州工业园区唯可达生物科技有限公司提供。用NaHCO 3缓冲液(pH 9.6)包被蛋白，4℃过夜，包被浓度为10μg/mL。用含0.1％牛血清白蛋白(bovine serum albumin,BSA)的磷酸盐缓冲液(PBS)37℃封闭30分钟，再用含0.5％吐温20的磷酸盐缓冲液(PBST)洗5遍后，室温孵育小鼠血清1小时，初始稀释度为1:100，PBST洗5遍后，1:5000孵育羊抗鼠HRP二抗(美国Santacruz公司)，37℃、30分钟，PBST洗5遍后用3,3,5,5-四甲基联苯胺(3,3,5,5-tetraamthyl benzidine,TMB)底物显色，37℃、15分钟，用2M稀硫酸终止后用酶标仪(美国Thermo公司)在450nm波长下读取吸光度(A)值。大于阴性对照A值2.1倍判断为阳性值，阳性值中的最高稀释度的倒数定义为血清抗体滴度。滴度小于起始稀释度1:100时，定义其滴度为50。
表23：分组与免疫规划
Figure PCTCN2019090877-appb-000007
Figure PCTCN2019090877-appb-000008
细胞免疫应答检测结果如图26所示。其中，初免pVKD1.0-CI-LMNB DNA疫苗，LMNB-C5蛋白加强(即实施例11中的C组)和LMNB-C10蛋白加强(即实施例11中的D组)免疫效果最好，显著性高于其平行对照(B组)。表明装有CMV病毒5个Th表位(C组)和10个Th表位(D组)均能大幅度提高弱免疫原的细胞免疫应答水平。
实施例13肿瘤预防动物实验
实施例2、3以及实施例8所制备的疫苗信息如表19。DNA疫苗载体pVKD1.0由苏州工业园区唯可达公司提供，DNA疫苗pVKD1.0-NP(表达源于毒株A/Shanghai/02/2013(H7N9))的流感抗原NP(NCBI参考序列:YP_009118476.1)由苏州工业园区唯可达生物科技有限公司提供，蛋白疫苗VP1(肠道病毒71型VP1蛋白，参见中国专利申请201310088364.5)由苏州工业园区唯可达生物科技有限公司提供。
从苏州大学动物实验中心购买60只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表24。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种4T1-hNY-ESO-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm 3)＝长×宽 2/2。当小鼠肿瘤体积超过2000mm 3时，对小鼠处死。
表24：分组与免疫规划
Figure PCTCN2019090877-appb-000009
Figure PCTCN2019090877-appb-000010
各组免疫小鼠肿瘤生长情况如图27所示。其中，对照组(A组)小鼠在攻瘤后(即肿瘤接种后)第14天全部出现肿瘤，并迅速生长。各免疫组小鼠肿瘤生长滞后于对照组，其中，LMNB-I13加强免疫组(D组)小鼠和LMNB-I13与LMNB-C10混合加强免疫组(E组)小鼠肿瘤生长最慢，因此，此两组疫苗效果最好。
此外，小鼠无瘤存活分析的结果如图28所示。其中，对照组A组小鼠的无瘤存活(TFS)中位数为14天。各疫苗免疫组小鼠无瘤存活均显著高于对照组。表明所有疫苗均能提高免疫后小鼠的无瘤存活期。其中，带有I13表位融合肽的D组，带有I13和C10表位融合肽的E组和F组效果最好，最高增加了一倍的小鼠无瘤存活期。与常规疫苗组(B组)相比，带有I13表位融合肽的疫苗组(D组)显著提高了无瘤存活期，提高了约40％的无瘤存活。表明装有流感病毒13个Th表位或10个CMV Th表位能大幅提高肿瘤疫苗的肿瘤保护效果。
最后，小鼠总体存活分析的结果如图29所示。其中，对照组A组小鼠的总体存活(OS)中位数为35天。各疫苗免疫组小鼠总体存活均显著高于对照组。表明所有疫苗均能提高免疫后小鼠的存活。其中，带有I13表位融合肽的D组，带有I13和C10表位融合肽的E组和F组效果最好，总体存活最高增加了83％。与常规疫苗组(B组)相比，带有I13表位融合肽的疫苗组(D组和F组)显著增加了无瘤存活期，最高增加了28％。表明装有流感病毒13个Th表位或10个CMV Th表位能大幅提高肿瘤疫苗的肿瘤保护效果。
实施例14肿瘤治疗实验
所涉及的疫苗见实施例9。从苏州大学动物实验中心购买30只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表25。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种肿瘤细胞4T1-hNY-ESO-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，分别在肿瘤细胞接种后第1天，第8天和第15天给相应小鼠皮下接种蛋白疫苗。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm 3)＝长×宽 2/2。当小鼠肿瘤体积超过2000mm 3时，对小鼠处死。
表25：分组与免疫规划
Figure PCTCN2019090877-appb-000011
各组免疫小鼠肿瘤生长情况如图30所示。其中，对照组(A组)小鼠在攻瘤后(即肿瘤接种后)第14天全部出现肿瘤，并迅速生长。与未治疗的对照组(A组)相比，LMNB-I13蛋白疫苗治疗组(C组)小鼠肿瘤生长最慢。而且，在小鼠攻瘤后第22天，LMNB-I13蛋白疫苗治疗组小鼠肿瘤大小显著小于对照组(A组)，直到第30天，两组之间的肿瘤大小还存在显著性差异。到第35天，C组小鼠肿瘤生长开始加快，可能与LMNB-I13蛋白疫苗停止接种有关。该结果表明LMNB-I13蛋白疫苗能抑制小鼠肿瘤生长。
实施例15肿瘤治疗实验
所涉及的疫苗见实施例9。从苏州大学动物实验中心购买30只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表26。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种肿瘤细胞CT26-hLAGE-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，分别在肿瘤细胞 接种后第1天，第8天和第15天给相应小鼠皮下接种蛋白疫苗。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm 3)＝长×宽 2/2。当小鼠肿瘤体积超过2000mm 3时，对小鼠处死。
表26：分组与免疫规划
Figure PCTCN2019090877-appb-000012
各组免疫小鼠肿瘤生长情况如图31所示，由于未治疗对照组(A组)小鼠在攻瘤后(即肿瘤接种后)有小鼠未能成功接种肿瘤，故未纳入分析，比较了平行疫苗对照组(B组)和LMNB-I13治疗组(C组)小鼠。与B相比，C组小鼠肿瘤生长较慢，而且在在小鼠攻瘤后第22天，LMNB-I13蛋白疫苗治疗组小鼠肿瘤大小显著小于平行疫苗对照组(B组)，直到第30天，两组之间的肿瘤大小还存在显著性差异。同样地，在CT26小鼠模型也观察到C组小鼠在后期肿瘤生长开始加快，可能与LMNB-I13蛋白疫苗停止接种有关。该结果表明LMNB-I13蛋白疫苗能抑制小鼠肿瘤生长。
实施例16穿梭载体pSC65-LMNB-I13构建
I13氨基酸序列(SEQ ID NO:60)含有表4中的HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221在内的8个流感病毒Th表位和包括表4中的HA434、HA440、NP324、M1-127和M1-210在内的5个流感病毒Th表位。编码I13氨基酸序列的真核密码子优化序列(SEQ ID NO:61)经苏州金唯智生物科技有限公司合成后，通过本领域熟知的分子生物学方法插入穿梭载体pSC65(addgene，货号：30327)上，构建成含有13个流感表位的穿梭载体pSC65-I13。
表达三联融合肿瘤抗原LAGE-1、MAGE-A3、NY-ESO-1以及霍乱毒素B亚单位的载体pVKD1.0-hLMN-CTB(见实施例2)。然后将所述载体中的LMN-CTB片段通过Sal I与Kpn I酶切回收后插入前述的穿梭载体pSC65-I13上，构建成表达三联肿瘤抗原的穿梭载体pSC65-LMNB-I13(质粒图谱如图32)，经测序鉴定正确后入库。其中，LMNB-I13的氨基酸序列如SEQ ID NO:62所示，其真核密码子优化序列如SEQ ID NO:63所示。用限制内切酶Sal I 与Kpn I鉴定载体pSC65-LMNB-I13(酶切体系如表27)，其酶切验证图谱如图33所示。
表27质粒pSC65V3的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pSC65-LMNB-I13 3μL，约1μg
Sal I(宝生物，货号1080A) 1μL
Not I(宝生物，货号1166A) 1μL
酶切缓冲液 1μL
ddH2O 补至10μL
实施例17重组痘苗病毒载体rvv-LMNB-I13构建
在143B细胞中获得重组痘苗病毒载体，具体方法如下。第1天，在6孔细胞培养板(JET，TCP-010-006)铺143B细胞(苏州工业园区唯可达生物科技有限公司提供)，1×10 6/孔，于37℃二氧化碳细胞培养箱中过夜孵育。第二天，以0.05 MOI(即5×10 4 PFU(空斑形成单位)/孔)加入痘苗病毒野生株(由北京生物制品所提供)，然后置于37℃二氧化碳细胞培养箱中孵育两个小时，期间准备穿梭载体/转染试剂复合物。其中穿梭载体为实施例16中获得的pSC65-LMNB-I13，转染试剂为Turbofect(Thermo Fisher Scientific，R0531)，转染剂量与复合方法可参见转染试剂说明书。复合体系完成后，将143B细胞上清换为2mL/孔的含2％胎牛血清(FBS)的DMEM维持培养基，然后加入穿梭载体/转染试剂复合物。转染48小时后，去上清，收集细胞，并在0.5mL维持培养基中重悬，反复冻融三次，然后将重组细胞裂解物接入新的143B细胞上(含50μg/mL BrdU)，37℃孵育1到2天。期间观察细胞病变，待病毒噬斑出现合适数量时(低于20空斑/孔)，进行单斑纯化。
单斑纯化
将2％低熔点琼脂糖微波炉加热(中高火2分钟左右)至沸，转移至45℃水浴降温并防止其凝固。
吸取适量2×维持培养液(1mL/孔)，按体积比1：50加入X-gal储存液，在45℃水浴中预热。
按等体积比例混合低熔点琼脂糖与含X-gal的2×维持培养液，制成铺斑固体培养基，去细胞上清，小心加入铺斑固体培养基，然后转移至4℃冰箱凝固10分钟，期间不要动6孔板，防止凝固不均匀。
待完全凝固后，转移6孔板至37℃细胞培养箱中孵育2至4小时(有时过夜)，直至蓝斑出现。
待蓝斑出现后，用1mL枪头(预先用剪刀将枪头剪平)优先挑取分散较 好的，颜色较深的蓝斑，挑取时一定要将固体培养基下面的细胞层挑到，每孔挑取若干个蓝斑，分别转移至含0.5mL维持培养液的Ep管中。
振荡混匀含病毒的Ep管，反复冻融三次(-80℃冰箱约5分钟，室温约2分钟)，最后振荡混匀，-80℃冻存。
重复六轮单斑纯化，直至纯度至100％。
实施例18重组痘苗病毒载体rvv-LMNB-I13扩增制备与滴定
将实施例17中构建的重组痘苗病毒载体rvv-LMNB-I13，以及痘苗病毒野生株分别在Vero细胞(苏州工业园区唯可达生物科技有限公司提供)上扩增，扩增方法如下。
前一天，准备汇集度100％的Vero单层细胞(1×10 7细胞/皿)，共10皿。
去上清，换为维持培养基，将待扩增的痘病毒接种到细胞上(0.01PFU/细胞)，37℃培养箱孵育2-3天，观察可见明显的细胞病变。
将细胞刮下并收集，1800g离心5分钟，去上清。
用5mL维持培养基进行重悬，在冰上用超声波细胞粉粹机超声，超声条件为：50瓦，5秒超声/5秒间隔，共15分钟。
反复冻融两次(-80℃冰箱约5分钟，室温约2分钟)，最后振荡混匀；
在二级生物安全柜中进行分装至1.5mL离心管中，1mL/支，-80℃冻存。
扩增制备好的痘苗病毒在Vero细胞上进行感染效价滴定，具体方法如下。
前一天，在24孔板中，准备汇集度100％的Vero细胞，3×10 5/孔。
去上清，每孔添加200μL维持培养液，以防止细胞干涸。
取100μL待测痘病毒加入900μL维持培养基，十倍稀释，连续稀释10 1，10 2，10 3，直到10 9倍。注意：进行稀释时，因为由高浓度向低浓度稀释，每次向低浓度稀释应更换枪头。
从病毒浓度由小到大(10 9，10 8，……10 4)添加到24孔板中，每孔400μL稀释液，两个重复，连续测定6个稀释倍数。将添加完的24孔板放入37℃细胞培养箱中孵育2天。
显微镜下数出病毒蚀斑的数目，多于20的，记为20+。将可以数出的20(含)以内的蚀斑数目两复孔求平均×2.5(1000μL/400μL)×相应孔的稀释倍数，即为重组病毒滴度(PFU/mL)。
痘苗病毒载体效价滴定结果如表28所示。
表28痘苗病毒载体效价滴定
痘苗病毒 效价(PFU/mL)
痘苗病毒野生型rvv-wt 1.5×10 8
重组痘苗病毒rvv-LMNB-I13 1.0×10 8
实施例19肿瘤治疗实验1
从苏州大学动物实验中心购买20只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。在第0天，所有小鼠皮下接种表达MAGE-A3肿瘤抗原的肿瘤细胞CT26-MAGE-A3稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为2×10 5细胞/只，然后随机分成2两组。在肿瘤细胞接种后第1天，第14天和第28天给相应小鼠小腿胫骨前肌接种实施例18中制备的痘苗病毒载体(具体疫苗接种规划如表29)。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm 3)＝长×宽 2/2。当小鼠肿瘤体积超过10000mm 3时，对小鼠处死。
表29实验动物分组与疫苗接种规划
Figure PCTCN2019090877-appb-000013
各组免疫小鼠肿瘤生长情况如图34所示。其中，对照组小鼠在肿瘤接种后第15天全部出现肿瘤，并迅速生长。在小鼠攻瘤后第30天，治疗组小鼠肿瘤平均大小显著小于对照组。结果表明痘苗病毒载体疫苗rvv-LMNB-I13能抑制带有MAGE-A3表达的肿瘤生长。
小鼠存活状况的生存曲线结果如图35所示，痘苗病毒载体疫苗rvv-LMNB-I13治疗组小鼠总体生存(OS中位数36天)优于对照组小鼠(OS中位数33天，p<0.05)。结果表明痘苗病毒载体疫苗rvv-LMNB-I13能提高患有表达MAGE-A3肿瘤的小鼠生存。
实施例20肿瘤治疗实验2
从苏州大学动物实验中心购买20只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。在第0天，所有小鼠皮下接种表达LAGE-1肿瘤抗原的肿瘤细胞CT26-LAGE-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，然后随机分 成2两组。在肿瘤细胞接种后第1天，第14天和第28天给相应小鼠小腿胫骨前肌接种实施例18中制备的痘苗病毒载体(具体疫苗接种规划如表30)。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm 3)＝长×宽 2/2。当小鼠肿瘤体积超过10000mm 3时，对小鼠处死。
表30实验动物分组与疫苗接种规划
Figure PCTCN2019090877-appb-000014
各组免疫小鼠肿瘤生长情况如图36所示。在攻瘤后第26天，对照组小鼠肿瘤生长迅速，并显著大于治疗组小鼠，一直持续到第50天。结果表明痘苗病毒载体疫苗rvv-LMNB-I13能抑制带有LAGE-1表达的肿瘤生长。
此外，小鼠存活状况的生存曲线结果如图37所示，痘苗病毒载体疫苗rvv-LMNB-I13治疗组小鼠总体生存(OS中位数75天)优于对照组小鼠(OS中位数51天，p<0.05)。结果表明痘苗病毒载体疫苗rvv-LMNB-I13能提高患有表达LAGE-1肿瘤的小鼠生存。
将实施例13-15中蛋白疫苗的免疫效果与实施例19-20中重组痘苗病毒载体的治疗效果相比，蛋白疫苗在预防肿瘤效果显著(参见图27、28、29)，虽然蛋白疫苗在肿瘤治疗上能维持一定的疗效，但不算特别明显，也不能持续(参见图30、31)。而重组痘苗病毒载体在肿瘤治疗上取得了特别显著的效果，不但持续抑制了肿瘤生长，还在总体存活了有明显改善(参见图34-37)，效果显著。

Claims (19)

  1. 一种重组病毒载体，所述重组病毒载体包含编码CD4辅助性T细胞表位融合肽的多核苷酸，所述CD4辅助性T细胞表位融合肽包含巨细胞病毒表位和/或流感病毒表位。
  2. 根据权利要求1所述的重组病毒载体，所述表位融合肽包含选自SEQ ID NO:1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO:11-23所示的流感病毒表位中的一种或多种。
  3. 根据权利要求1或2所述的重组病毒载体，所述表位融合肽由5个或10个巨细胞病毒表位和/或由8个或13个流感病毒表位组成，例如SEQ ID NO:34或44所示的表位融合肽；
    优选地，所述表位融合肽由13个流感病毒表位组成，例如SEQ ID NO:48或60所示的表位融合肽。
  4. 根据权利要求1-3任一项所述的重组病毒载体，所述表位融合肽诱导体液或细胞免疫应答。
  5. 根据权利要求1-4任一项所述的重组病毒载体，其中所述重组病毒是痘苗病毒载体，优选为复制型痘苗病毒载体，例如痘苗病毒天坛株，例如752-1株，或者为非复制型痘苗病毒载体，例如痘苗病毒减毒疫苗安卡拉株(Modified Vaccinia Ankara，MVA)。
  6. 根据权利要求1-5任一项所述的重组病毒载体，其还包含编码目的免疫原的多核苷酸；优选地，所述重组病毒载体包含编码所述表位融合肽和目的免疫原的融合蛋白的多核苷酸。
  7. 根据权利要求6所述的重组病毒载体，其中，所述目的免疫原选自肽、抗原、半抗原、碳水化合物、蛋白质、核酸、过敏原、病毒或病毒的一部分、细菌、寄生虫或其它完整的微生物；优选地，所述抗原为肿瘤抗原或感染相关抗原；进一步优选地，所述肿瘤抗原选自肺癌抗原、睾丸癌抗原、黑色素瘤抗原、肝癌抗原、乳腺癌抗原或前列腺癌抗原中的一种或多种；优选地，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种；进一步优选地，所述LAGE抗原为LAGE-1，所述MAGE抗原为MAGE-A3；优选地，所述LAGE-1的氨基酸序列如SEQ ID NO:24所示，所述MAGE-A3的氨基酸序列如SEQ ID NO:25所示，所述NY-ESO-1的氨基酸序列如SEQ ID NO:26所示；还进一步优选地，所述肿瘤抗原包含LAGE-1、MAGE-A3和NY-ESO-1；优选地，所述感染相关抗原选自HIV抗原、流感病毒抗原或HBV抗原中的一种或多种；优选地，所述融合蛋白的 氨基酸序列如SEQ ID NO:55-58和62中之一所示，优选为SEQ ID NO:62；优选地，编码所述融合蛋白的多核苷酸序列如SEQ ID NO:63所示。
  8. 一种CD4辅助性T细胞的表位融合肽，其具有SEQ ID NO:60所示的氨基酸序列。
  9. 一种融合蛋白，其包含根据权利要求8所述的表位融合肽和目的免疫原；优选地，所述融合蛋白具有如SEQ ID NO:62所示的氨基酸序列。
  10. 根据权利要求9所述的融合蛋白，其中，所述目的免疫原选自肽、抗原、半抗原、碳水化合物、蛋白质、核酸、过敏原、病毒或病毒的一部分、细菌、寄生虫或其它完整的微生物；优选地，所述抗原为肿瘤抗原或感染相关抗原；进一步优选地，所述肿瘤抗原选自肺癌抗原、睾丸癌抗原、黑色素瘤抗原、肝癌抗原、乳腺癌抗原或前列腺癌抗原中的一种或多种；优选地，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种；进一步优选地，所述LAGE抗原为LAGE-1，所述MAGE抗原为MAGE-A3；优选地，所述LAGE-1的氨基酸序列如SEQ ID NO:24所示，所述MAGE-A3的氨基酸序列如SEQ ID NO:25所示，所述NY-ESO-1的氨基酸序列如SEQ ID NO:26所示；还进一步优选地，所述肿瘤抗原包含LAGE-1、MAGE-A3和NY-ESO-1；优选地，所述感染相关抗原选自HIV抗原、流感病毒抗原或HBV抗原中的一种或多种。
  11. 一种多核苷酸，其编码根据权利要求8所述表位融合肽或根据权利要求9或10所述的融合蛋白；优选地，编码所述表位融合肽的多核苷酸序列如SEQ ID NO:61所示；优选地，编码所述融合蛋白的多核苷酸序列如SEQ ID NO:63所示。
  12. 一种免疫组合物，其包含治疗有效量的根据权利要求1-7中任一项所述的重组病毒载体、权利要求8所述的表位融合肽、权利要求或9或10所述的融合蛋白和/或权利要求11所述的多核苷酸，以及药学上可接受的载体。
  13. 根据权利要求12的免疫组合物，其为疫苗。
  14. 一种药盒，其包含根据权利要求1-7中任一项所述的重组病毒载体、根据权利要求8所述的表位融合肽、根据权利要求或9或10所述的融合蛋白、根据权利要求11所述的多核苷酸和/或根据权利要求12或13所述的免疫组合物，以及其使用说明。
  15. 根据权利要求1-7中任一项所述的重组病毒载体、根据权利要求8所述的表位融合肽、根据权利要求或9或10所述的融合蛋白、根据权利要求11所述的多核苷酸和/或根据权利要求12或13所述的免疫组合物在制备 提高目的免疫原的免疫原性的药物中的用途。
  16. 根据权利要求1-7中任一项所述的重组病毒载体、根据权利要求8所述的表位融合肽、根据权利要求或9或10所述的融合蛋白、根据权利要求11所述的多核苷酸和/或根据权利要求12或13所述的免疫组合物在制备提高目的免疫原的免疫原性的疫苗中的用途。
  17. 根据权利要求1-7中任一项所述的重组病毒载体、根据权利要求8所述的表位融合肽、根据权利要求或9或10所述的融合蛋白、根据权利要求11所述的多核苷酸和/或根据权利要求12或13所述的免疫组合物在制备治疗或预防有需要的受试者的病症的药物中的用途。
  18. 根据权利要求17所述的用途，所述的病症选自恶性肿瘤、细菌和病毒性感染中的一种或多种；优选地，所述感染是慢性感染。
  19. 根据权利要求18所述的用途，所述恶性肿瘤为乳腺癌或结肠癌。
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