WO2022100459A1 - 一种预防和治疗默克尔细胞癌的新型疫苗 - Google Patents

一种预防和治疗默克尔细胞癌的新型疫苗 Download PDF

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WO2022100459A1
WO2022100459A1 PCT/CN2021/127386 CN2021127386W WO2022100459A1 WO 2022100459 A1 WO2022100459 A1 WO 2022100459A1 CN 2021127386 W CN2021127386 W CN 2021127386W WO 2022100459 A1 WO2022100459 A1 WO 2022100459A1
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vaccine
seq
cells
protein
tumor
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French (fr)
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何悦
赵干
张璐楠
程鑫
俞庆龄
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艾棣维欣(苏州)生物制药有限公司
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Priority to US18/253,034 priority Critical patent/US20230414742A1/en
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Definitions

  • the invention belongs to the fields of immunology and virology, in particular to a novel vaccine for preventing and treating Merkel cell carcinoma.
  • Merkel cell carcinoma polyomavirus is currently the only polyomavirus known to be directly associated with human cancer.
  • Merkel cell carcinoma polyoma virus is a double-stranded non-enveloped DNA virus with an icosahedral spherical structure of about 45 nm and a genome size of 5386 bp, containing early coding regions, late coding regions and non-coding regulatory regions ( NCRR).
  • NCRR non-coding regulatory regions
  • the early genes of Merkel cell carcinoma polyomavirus mainly encode LT, ST, 57kT and ALTO (alternating frame of large T open reading frame), while the late genes mainly encode the capsid proteins VP1 and VP2.
  • VP1 has the function of initiating viral infection and often exists in the form of pentamers, and 72 pentamers can self-assemble into virus-like particles in eukaryotic systems.
  • Merkel cell carcinoma is a rare, aggressive form of skin cancer that appears as fast-growing purple-red nodules on areas such as the head and neck that are exposed to sunlight.
  • Merkel cell carcinoma polyomavirus integrated into tumor cells can be detected in approximately 80% of Merkel cell carcinoma (MCC) patients' tumors.
  • MCC Merkel cell carcinoma
  • about 60%-80% of healthy people can detect the specific antibody of Merkel cell carcinoma polyoma virus capsid protein VP1.
  • Merkel cell carcinoma polyomavirus mainly infects human in infancy in an asymptomatic manner, and the weakened immunity caused by ultraviolet radiation, disease or aging is the inducement of Merkel cell carcinoma.
  • the incidence of Merkel cell carcinoma is not high, but its clinical prognosis is extremely poor, and it is one of the most lethal skin-derived tumors.
  • VLP virus-like particle
  • virus-like particle vaccines can treat established papilloma without any adjuvant.
  • Merkel cell carcinoma polyoma virus VP1 which can self-assemble into virus-like particles, the development of related vaccines based on this protein has shown great potential in the prevention or treatment of Merkel cell cancer. .
  • Chinese patent 200810232479.6 discloses a B19 virus VP1 unique region gene, and constructs a B19 prokaryotic expression pQE30-VP1 unique region cloning plasmid, expresses and purifies a recombinant protein, which can induce immune cell response in vitro and produce high titer of antibodies.
  • Chinese Patent No. 201010126145.8 discloses a codon-optimized EV71VP1 gene and its nucleic acid vaccine.
  • the vaccine can be effectively expressed in eukaryotic 293T cells, and immunized animals can stimulate the production of specific VP1 antibodies.
  • the vaccine only uses nucleic acid as the active ingredient of the vaccine, and the immune effect is poor.
  • the term "vaccine” as used herein refers to a composition that can be administered to a human or animal to induce an immune system response; such an immune system response can lead to the production of antibodies or to activate only certain cells, particularly antigen presenting cells, T lymphocytes cells and B lymphocytes.
  • the vaccine composition may be a composition for prophylactic purposes or for therapeutic purposes, or a composition for both prophylactic and therapeutic purposes.
  • administration refers to their usual and common meaning in the art of treating a patient with a vaccine or a certain composition.
  • co-administration and “concomitant administration” are synonymous and refer to the administration of two substances or two compositions to a patient in such a manner that both substances or both compositions are present in the patient's body.
  • co-administration may be simultaneous or sequential, and the co-administered substances or compositions may be administered to the patient at the same time, or separately but close in time, or on the same day, or in a manner that otherwise enables the individual substances or compositions in the body The dwell times were administered to patients in a manner that significantly overlapped.
  • Administration parenterally, may include subcutaneous, intramuscular, intradermal, intraperitoneal, intraocular, intranasal and intravenous.
  • the vaccines or compositions contemplated by the present invention can be administered to an individual according to methods known in the art. These methods include parenteral administration, for example by dermal injection or all routes of injection into the skin: eg, intramuscular, intravenous, intraperitoneal, intradermal, mucosal, submucosal or subcutaneous, and the like.
  • the vaccine may be administered topically as drops, sprays, gels or ointments to the mucosal epithelium of the eye, nose, mouth, anus or vagina, or to the outer epidermis anywhere on the body.
  • Other possible routes of administration are inhalation of sprays, aerosols or powders through the respiratory tract.
  • the particle size used will determine how deeply the particles enter the respiratory tract.
  • it may be administered by the digestive route, in the form of powder, liquid or tablet in combination with food, feed or drinking water, or directly to the oral cavity in the form of a liquid, gel, tablet or capsule, or in the form of a suppository in the anus.
  • the DNA vaccine can be delivered by using an electrical pulser or any electroporation device to facilitate infiltration of the DNA vaccine into host cells and efficient expression.
  • targeting refers to "treatment”, “prophylaxis”, “adjunctive therapy” or equivalent words and combinations thereof in the field of vaccines.
  • the compositions or methods of the present invention can provide any dose, any level of disease treatment or prevention in a mammal.
  • the treatment or prevention provided by the methods of the present invention may include treatment or prevention of one or more conditions or symptoms of a disease, cancer.
  • prevention may include delaying the onset of a disease or a symptom or disorder thereof.
  • the cancer can be any cancer, including any cancer associated with any of the tumor antigens described herein.
  • adjuvant means that when used in combination with a particular immunogen in a formulation (eg, a VLP-based vaccine), an immune response will be altered or modified in an enhanced or reduced manner. Modifications of the immune response include enhancing or amplifying a specific antibody or cellular immune response, either or both are included within the scope. Modification of an immune response can also refer to the reduction or inhibition of certain antigen-specific immune responses.
  • virus-like particle refers to a non-replicating viral capsid particle.
  • viral structural proteins such as envelope or capsid proteins
  • VLPs are typically composed of one or more viral proteins, such as, but not limited to, those proteins known as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins.
  • VLPs can form spontaneously after recombinant expression of the protein in an appropriate expression system. Methods for generating specific VLPs are known in the art and are discussed more fully below.
  • VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, Baker et al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol. (1994) 68:4503-4505.
  • VLPs can be isolated by density gradient centrifugation and/or by characteristic density bands.
  • vitrified aqueous samples of the VLP formulations in question can be examined using cryo-electron microscopy and images recorded under appropriate exposure conditions.
  • Other methods of VLP purification include, but are not limited to, chromatographic techniques such as affinity, ion exchange, size exclusion and reversed-phase processes.
  • any nanometer measurement device can be used to measure the size of nanoparticles.
  • the present invention also relates to variants of polynucleotides and polypeptides.
  • variant refers to a polynucleotide or polypeptide that differs from the polynucleotide or polypeptide of the present invention but retains its essential properties.
  • variants are generally very similar and, in many regions, identical to the polynucleotides or polypeptides of the invention.
  • Variants may contain changes in either or both coding regions, noncoding regions. Most tend to produce silent substitutions, additions or deletions, but do not alter the properties or activities of the encoded polypeptide.
  • Polynucleotide variants are preferred.
  • nucleotide variants resulting from silent substitutions due to the degeneracy of the genetic code are preferred.
  • 5-10, 1-5 or 1-2 amino acids are substituted in any combination, and deletion or addition variants are also common.
  • An example of a variant is a truncation
  • an example of a truncation is an MCV-VP1 truncation that can include a C-terminal 55aa(369-423) deletion.
  • C'-deletion from wild-type MCV-VP1 can generate MCV-VP1 truncations with the aa1-369 fragment, which can be used with other antigens as fusion proteins for chimeric VLPs.
  • An example of a variant is a deletion, and an example of a deletion is the C-terminal 17aa (352-368) deletion of MCV-VP1.
  • An example of a variant is a mutation
  • an example of a mutation is the E353 mutation of MCV-VP1, replaced by L or F.
  • the present invention also includes allelic variants of the polynucleotides.
  • An allelic variant represents any one of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variants arise naturally through mutation and can lead to polymorphisms within a population. Genetic mutations can be silent (no change in the encoded polypeptide) or can encode a polypeptide with an altered amino acid sequence.
  • An allelic variant of a polypeptide is the polypeptide encoded by the allelic variant.
  • the present invention also relates to polynucleotide fragments comprising or hybridizing to non-coding regions of MCV VP1.
  • a polynucleotide fragment can be a polynucleotide fragment of equal or shorter length than naturally occurring nucleotides. Such fragments can be used for diagnosis. Fragments of polynucleotides that contain cleavage points or hybridize to polynucleotides that contain cleavage points frequently occur. Polynucleotide fragments of any length such as 50, 150, 500, 600, 1000, 1100 or about 1260 nucleotides can be used.
  • the optimized cDNA sequence of Merkel cell carcinoma polyoma virus (MCV) structural protein VP1 is cloned in eukaryotic system and prokaryotic system, and VP1 protein is expressed in prokaryotic system to construct a VP1-based protein targeting Merck DNA and/or protein vaccines against cell carcinoma polyomavirus.
  • the DNA vaccine provided by the present invention alone against Merkel cell carcinoma or the protein vaccine against Merkel cell carcinoma can induce cellular immune response or high level of anti-VP1 antibody.
  • This study is the first to use VP1 as a vaccine antigen against Merkel cell carcinoma polyoma virus, laying the foundation for vaccine research against Merkel cell carcinoma.
  • Vehicle herein refers to "PBS”
  • OVA refers to "ovalbumin”.
  • VP1 or “recombinant protein VP1” herein refers to “recombinant Merkel cell carcinoma polyoma virus capsid protein”
  • rVP1 or “reassembled or self-assembled VP1” refers to “recombined or self-assembled VP1”.
  • Self-assembling Merkel cell carcinoma polyoma virus capsid proteins
  • the present invention provides a vaccine composition against Merkel cell carcinoma, characterized in that, the vaccine composition comprises Merkel cell carcinoma polyoma virus capsid protein VP1 or contains a protein encoding Merkel cell carcinoma A plasmid for the gene for the polyoma capsid protein VP1, and a TLR agonist selected from one or more of CpG, R848, and MPL.
  • the one comprising Merkel cell carcinoma polyoma virus capsid protein VP1 is a protein vaccine composition.
  • the plasmid containing the gene encoding the Merkel cell carcinoma polyoma virus capsid protein VP1 is a DNA vaccine composition.
  • the TLR agonist is a combination of CpG and R848.
  • the composition further comprises an aluminum adjuvant.
  • the vaccine comprises 5-500 parts by weight of Merkel cell carcinoma polyomavirus capsid protein VP1 protein or a gene encoding Merkel cell carcinoma polyomavirus capsid protein VP1 Plasmid, 2.5-250 parts by weight of TLR agonist and 10-1000 parts by weight of aluminum adjuvant.
  • the vaccine comprises 10-100 parts by weight of Merkel cell carcinoma polyomavirus capsid protein VP1 protein or a gene encoding Merkel cell carcinoma polyomavirus capsid protein VP1 Plasmid, 5-100 parts by weight of TLR agonist and 50-500 parts by weight of aluminum adjuvant.
  • the nucleotide sequence of VP1 has 71.9-74.7% homology with SEQ ID No.6.
  • nucleotide sequence of the VP1 is shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4.
  • the present invention provides a gene encoding Merkel cell carcinoma polyoma virus capsid protein VP1.
  • the coding gene has a nucleotide sequence of 71.9-74.7% homology with SEQ ID No.6.
  • the encoding gene is the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4.
  • the present invention also provides the use of the above-mentioned Merkel cell carcinoma polyoma virus capsid protein VP1 encoding gene in preparing a vaccine against Merkel cell carcinoma.
  • the application is to prepare a DNA vaccine against Merkel cell carcinoma using one or more of the nucleotide sequences with 71.9-74.7% homology to SEQ ID No. 6 or variants thereof.
  • the application is to use one or more of the nucleotide sequences shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or their variants Preparation of DNA vaccines against Merkel cell carcinoma.
  • the application is to utilize one or more of the proteins encoded by the nucleotide sequence having 71.9-74.7% homology with SEQ ID No. 6 and/or its variants and/or its mutants Preparation of a protein vaccine against Merkel cell carcinoma.
  • the application is to utilize the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 and/or the protein encoded by its variant and/or One or more of its mutants are used to prepare a protein vaccine against Merkel cell carcinoma; further specifically, the protein includes, but is not limited to, the amino acid sequence of SEQ ID No.5.
  • the application is to simultaneously utilize a nucleotide sequence having 71.9-74.7% homology with SEQ ID No. 6 and/or its variability and one of the encoded proteins and/or its mutants or more to prepare a vaccine against Merkel cell carcinoma.
  • the application is to simultaneously utilize the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 and/or its variants and the above nucleosides
  • One or more of the proteins encoded by the acid sequences and/or mutants thereof prepare a vaccine against Merkel cell carcinoma.
  • the application includes, but is not limited to, the preparation of a vaccine against Merkel cell carcinoma using the amino acid sequence of SEQ ID No. 5.
  • the present invention provides a vaccine against Merkel cell carcinoma.
  • the vaccine is a DNA vaccine.
  • the DNA vaccine comprises one or more of nucleotide sequences and/or variants thereof having 71.9-74.7% homology with SEQ ID No. 6.
  • the DNA vaccine comprises one or more of the nucleotide sequences of SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 and/or variants thereof .
  • the DNA vaccine is constructed on a vector
  • the vector includes but is not limited to pVAX1 vector, pcDNA3.1, NTC8385, or viral vector, such as adenovirus Ad vector, adeno-associated virus AAV vector, and the like.
  • the protein vaccine comprises a nucleotide sequence with 71.9-74.7% homology to SEQ ID No. 6 and/or one or more of the proteins encoded by its variants and/or its mutants kind.
  • the protein vaccine comprises the protein encoded by SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 and/or its variant nucleotide sequence and/or its mutation one or more of the body.
  • the protein encoded by SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 includes but is not limited to the amino acid sequence of SEQ ID No.5.
  • the injection amount of the DNA vaccine is 1-200 ⁇ g/mouse.
  • the protein vaccine can be prepared by using E. coli system.
  • the VP1 protein is produced to form DNA-free virus-like particles (VLPs) of varying sizes, eg, by self-assembly.
  • VLPs DNA-free virus-like particles
  • the protein vaccine can be prepared by using protein expression systems of other organisms. Including but not limited to yeast expression system, baculovirus expression system, Chinese hamster ovary cell (CHO) expression system, etc.
  • the virus-like particles in the protein vaccine are purified before the protein vaccine is formed.
  • the size of VP1 protein virus-like particles is about 1 nm-about 1 ⁇ m, about 5 nm-about 800 nm, about 8 nm-about 700 nm, about 10 nm-about 400 nm, about 20 nm-about 200 nm , about 25nm-about 100nm, or between about 30nm-about 50nm; in one embodiment, the virus-like particle size of the VP1 protein is more concentrated in about 20nm-about 200nm, about 25nm-about 100nm, or about 30nm-about 50nm between.
  • the vaccine further comprises an adjuvant, the adjuvant comprising a TLR agonist and an aluminum adjuvant, the TLR agonist being selected from one or more of CpG, R848 and MPL.
  • the vaccine comprises 5-500 parts by weight of DNA vaccine or protein vaccine, 2.5-250 parts by weight of TLR agonist and 10-1000 parts by weight of aluminum adjuvant.
  • the vaccine comprises 10-100 parts by weight of DNA vaccine or protein vaccine, 5-100 parts by weight of TLR agonist and 50-500 parts by weight of aluminum adjuvant.
  • the injection amount of the protein vaccine is 1.1-20 ⁇ g/mouse.
  • the vaccines are prophylactic and/or therapeutic vaccines.
  • the vaccine provided by the present invention is applicable to any mammal.
  • Said mammals refer to warm-blooded vertebrates, including but not limited to humans, rabbits, cats, dogs, pigs, cattle and dogs.
  • the dosage forms of the vaccines or products containing the vaccines involved in the present invention include, but are not limited to, drops, sprays, gels, powders, ointments, tablets, and granules.
  • FIG. 1 is a graph showing the tumor growth curve of the CMS5-VP1 cell line in Basic Experimental Example 1.
  • FIG. 1 is a graph showing the tumor growth curve of the CMS5-VP1 cell line in Basic Experimental Example 1.
  • FIG. 2 is the detection result of the VP1 protein vaccine prepared in Example 3.
  • FIG. 2 is the detection result of the VP1 protein vaccine prepared in Example 3.
  • FIG. 3 is the result of SDS-PAGE detection of purified VP1 protein in Example 6.
  • FIG. 4 shows the results of Nu-PAGE in Example 7 to detect the VP1 structural proteins formed under different assembly processes.
  • Example 5 is the mouse-specific MCV-VP1 antibody titer, DTH (delayed type hypersensitivity), and cellular immunity results in the immunogenicity verification of the protein vaccine VP1 in Example 8.
  • FIG. 6 is the detection result of the immune response of the DNA vaccine in Example 9.
  • FIG. 7 shows the results of DTH and specific antibody titers 7 days after the combined immunization in Example 10.
  • Figure 8 is the tumor growth curve of tumor mice after tumor inoculation in Example 11, wherein the arrow position is the time point of vaccine immunization.
  • Figure 9 is a picture of tumor-bearing mice in each group on the 33rd day after tumor inoculation in Example 12.
  • Figure 10 is the tumor tissue of each group of tumor-bearing mice on the 33rd day after tumor inoculation in Example 12.
  • Figure 11 is the tumor growth curve of each group of tumor-bearing mice after tumor inoculation in Example 12.
  • FIG. 12 shows the degree of swelling of the soles of feet 24 hours after injection of rVP1 or PBS in Example 13.
  • FIG. 13 shows the experimental results on DTH in mice after the adjuvanted MCV-VP1 therapeutic tumor vaccine in Example 13.
  • FIG. 14 shows the VP1 antibody titer results of mice immunized with the adjuvanted therapeutic vaccine in Example 13.
  • FIG. 14 shows the VP1 antibody titer results of mice immunized with the adjuvanted therapeutic vaccine in Example 13.
  • FIG. 15 shows the results of flow cytometry of the expression levels of TNF- ⁇ , IFN- ⁇ and IL-2 in mouse CD4 + , CD8 + T cells immunized with the adjuvanted therapeutic vaccine in Example 13.
  • FIG. 15 shows the results of flow cytometry of the expression levels of TNF- ⁇ , IFN- ⁇ and IL-2 in mouse CD4 + , CD8 + T cells immunized with the adjuvanted therapeutic vaccine in Example 13.
  • FIG. 16 shows the tumor volume in each group of mice 19 and 34 days after tumor inoculation in Example 13.
  • Figure 17 shows the results of flow cytometry detection of TNF- ⁇ , IFN- ⁇ and IL-2 secretion levels of CD4 + , CD8 + T cells after mouse splenocytes were stimulated with VP1 19 days after tumor inoculation in Example 13.
  • Figure 18 shows the results of flow cytometry detection of TNF- ⁇ , IFN- ⁇ and IL-2 secretion levels of CD4 + and CD8 + T cells in mouse splenocytes stimulated with VP1 34 days after tumor inoculation in Example 13.
  • Figure 19 shows the antigen-specific antibody titers induced by the vaccine sVP1-VAC or rVP1-VAC prepared with the sVP1 or rVP1 antigen in Example 14.
  • FIG. 20 is the tumor growth curve of CMS5-VP1 in tumor-bearing mice in Example 15.
  • FIG. 20 is the tumor growth curve of CMS5-VP1 in tumor-bearing mice in Example 15.
  • FIG. 21 is the tumor growth curve of 4T1 tumor-bearing mice in Example 15.
  • FIG. 21 is the tumor growth curve of 4T1 tumor-bearing mice in Example 15.
  • Figure 22 shows the anti-tumor effects of the MCV therapeutic vaccine in Example 15 on three tumor models of CMS5-VP1, CMS5 and 4T1.
  • Figure 23 shows the tumor growth curve of mice immunized with MCV therapeutic vaccine in Experimental Example 17
  • the CMS5-VP1 tumor cell line will be mainly used for the verification of vaccine efficacy.
  • MCV-R (5'-GCGGCCGCTCTAGACTCGAGTCACAGCTCCTG-3').
  • the 5' end of MCV-F contains a Nhe I restriction site, and the 3' end of MCV-R contains a Not I restriction site.
  • telomere sequence containing an endonuclease site at the N-terminus and C-terminus was obtained by amplification.
  • the PCR reaction system is shown in Table 1 below.
  • the PCR-amplified VP1 fragment and the vector pCDH endonuclease NheI/Not I were digested in a 37°C water bath for 4 h, and then the VP1 fragment and the pcDH vector were recovered.
  • the enzyme digestion system is shown in Table 3 below.
  • the recovered VP1 and pcDH fragments were ligated with T4 ligase at room temperature for 40 minutes according to the molar mass ratio of 1:3.
  • the ligation product was transformed into E. coli clone host strain DH5 ⁇ competent cells by chemical transformation.
  • the specific method is:
  • PCR-positive clones were picked and cultured overnight at 37°C with shaking in 5 mL of LB liquid medium (50 ⁇ g/mL Amp).
  • the recovered VP1 fragment and the vector pcDH-GFP were digested with Nhe I/Not I, and the digested products were electrophoresed on a 1% agarose gel.
  • the gel band containing VP1 and pcDH was cut out, and the target fragment was recovered with a gel recovery kit (QIAGEN, 28115).
  • the recovered VP1 and pcDH were subjected to enzymatic ligation reaction according to the reaction system shown in Table 4.
  • the cells were transformed into E. coli DH5 ⁇ competent cells and labeled as pcDH-VP1/DH5 ⁇ . Subsequently, 10 monoclonal colonies were randomly picked for colony PCR. In order to further confirm the constructed pcDH-VP1, the positive clones were sequenced to identify the correct clones.
  • the pcDH-VP1/DH5 ⁇ glycerol bacteria were inoculated at 1:100 in 200 mL LB (50 ⁇ g/mL) liquid medium at 37°C for overnight shaking culture, and the extracted plasmid was used to construct the CMS5-VP1 cell line.
  • CMS5 cells are a drug-induced fibrosarcoma cell line in BALB/c mice.
  • CMS5 cells are adherent cells.
  • CMS5 cells themselves are not resistant to puromycin (Puromycin), and the Puromycin resistance gene carried by the vector pcDH is integrated into the cell genome to endow the cells with Puromycin resistance. Therefore, pcDH-VP1 was transfected into CMS5 cells by lipofection method. Under the pressure of Puromycin, CMS5 cells without pcDH-VP1 transfection died in large numbers, and the cell line CMS5-VP1 expressing VP1 was screened.
  • Puromycin puromycin
  • CMS5 cells were cultured in complete medium containing different concentrations of Puromycin in RPMI1640 (10% FBS, 1% double antibody). After 24 hours, trypan blue staining was used to observe the cell death rate under a microscope to determine the drug concentration of 100% lethality of CMS5 cells as the drug stress for CMS5-VP1 cell line screening.
  • the specific method is as follows:
  • CMS5 cells in good condition were digested with trypsin, and then prepared into a cell suspension with RPMI1640 complete medium.
  • the CMS5 cell suspension was plated in a 12-well cell culture plate at 1 ⁇ 10 6 /well, and cultured overnight in a 37° C. incubator with 5% CO 2 .
  • the medium was replaced with RPMI1640 complete medium containing 0.25 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, 2 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL and 20 ⁇ g/mL Puromycin.
  • trypsin-digested CMS5 cells were stained with trypan blue, and cell lethality was calculated under a microscope to determine the final Puromycin drug concentration.
  • the pcDH-VP1 plasmid was transfected into CMS5 cells by lipofection method. 48 hours after transfection, the cells with the target plasmid were screened by Puromycin drug pressure, and the stable expression cell lines were screened by long-term drug screening.
  • the specific method is as follows:
  • DNA (pcDH-VP1 or pcDH-GFP plasmid) and Lipofectamine2000 were respectively diluted in serum-free OPTI-MEM at a ratio of 1:3, and left at room temperature for 20 minutes.
  • CMS5-VP1 or CMS5-GFP cells were taken to extract RNA with a kit, and cDNA was obtained by RT-PCR amplification.
  • PCR amplification was performed with the amplified cDNA as template and MCV-F1 and MCV-R1 as primers.
  • PCR amplification results were detected by 1% agarose gel electrophoresis.
  • CMS5-VP1 cells were screened for monoclonal cells after 6 rounds of drug stress screening.
  • the specific method is as follows:
  • CMS5-VP1 cells were digested with trypsin, stained with trypan blue, and counted with a hemocytometer under a microscope.
  • the CMS5-VP1 cell line was obtained. 5 ⁇ 10 6 cells were collected by centrifugation at 1000 rpm for 5 minutes, and the expression of VP1 was detected by immunoblotting.
  • the specific method is as follows:
  • Anti-VP1 rabbit polyclonal antibody was diluted 1:5000 in 1 ⁇ TBST of 5% nonfat milk powder, the PVDF membrane was placed in the primary antibody diluent, and incubated at room temperature for 1 h.
  • ECL color development (Tianneng Company, 180-501): Mix the substrate reaction solutions 1 and 2 in a ratio of 1:1, put the PVDF membrane upside down in the ECL reaction solution, and detect by chemiluminescence.
  • CMS5 cells themselves are not resistant to Puromycin, and CMS5-VP1 transformed into pcDH-VP1 is resistant to Puromycin because the vector pcDH carries a Puromycin resistance gene.
  • Puromycin To determine the resistance concentration of CMS5 cells to Puromycin, they were cultured in RMPI1640 complete medium containing 0.25 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, 2 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL and 20 ⁇ g/mL Puromycin, respectively. CMS5 cells. After 24 hours of culture, the state of the cells was observed under a microscope. When the Puromycin concentration reached 5 ⁇ g/mL, the cells died massively.
  • Lipofectamine2000 take 10 ⁇ g of plasmid pcDH-VP1 or pcDH-GFP, and 30 ⁇ L of Lipofectamine2000 respectively diluted in 500 ⁇ L of serum-free OPTI-MEM. After standing for 5 minutes at room temperature, the diluted pcDH-VP1 and Lipofectamine 2000 are mixed. Let stand for 20 minutes.
  • the DNA-liposome complex formed by pcDH-VP1 or pcDH-GFP and Lipofectamine 2000 was added to the overnight cultured CMS5 cells. After mixing, the cells were incubated at 37°C in a 5% CO 2 incubator.
  • CMS5 was transfected with pcDH-VP1 carrying the reporter gene GFP as a positive control while constructing the CMS5-VP1 cell line.
  • pcDH-VP1 carrying the reporter gene GFP
  • the proliferation of CMS5-VP1 or CMS5-GFP cells was observed by fluorescence microscope, and the cells were observed under white light or GFP fluorescence, respectively.
  • PCR amplification was carried out with CMS5-VP1 or CMS5-GFP cDNA as template, MCV-F1 and MCV-R1 as primers, and plasmid pcDH-VP1 was used as positive control.
  • the amplified PCR product was detected by 1% agarose gel electrophoresis to detect the target gene VP1, and the size of the amplified band obtained from the CMS5-VP1 cDNA was consistent with the size of the positive control.
  • the 4th band is the negative control with CMS5 cDNA as the template, and the 5th band is the non-specific amplification result of CMS5-GFP.
  • the digested CMS5-VP1 cells were diluted and then subjected to two rounds of cell screening.
  • Five VP1-expressing CMS5-VP1 cell lines were identified by immunoblotting. Western blotting results showed that CMS5-VP1/E2 had the highest expression, followed by E4E5.
  • the cell lines CMS5-VP1/B5G2, F5 and E2 with low, medium and high expression levels were selected to carry out the next tumorigenic experiment.
  • the VP1 expression level of CMS5-VP1 cell line was determined by western blot, and 3 of them were selected for tumorigenic experiment.
  • the specific method is as follows:
  • CMS5-VP1 cells cultured in RPMI1640 complete medium Puromycin
  • CMS5 cells cultured in RPMI1640 complete medium were digested with trypsin respectively to prepare cell suspensions.
  • CMS5-VP1 cells were diluted to 1 ⁇ 10 7 /mL and 3 ⁇ 10 7 /mL with 1 ⁇ PBS, respectively, and CMS5 cells were diluted to 3 ⁇ 10 7 /mL.
  • BALB/c female mice aged 6-8 weeks were randomly divided into three groups. 1 ⁇ 10 6 or 3 ⁇ 10 6 CMS5-VP1 cells were subcutaneously injected into the back, respectively, and 3 ⁇ 10 6 CMS5 cells were subcutaneously injected into the back of the control group.
  • Tumor size was measured every other day starting from day 4 after tumor cell inoculation.
  • mice 6-8 week old BALB/c female mice were randomized into groups of three.
  • the CMS5-VP1 cells were inoculated subcutaneously, and the inoculation amount was 1 ⁇ 10 6 or 3 ⁇ 10 6 per mouse, and the inoculation amount of the positive control CMS5 cells was 3 ⁇ 10 6 .
  • tumor size was measured every two days. When the tumor grew to 2000mm 3 , the mice were euthanized, and the tumor tissue was collected to detect the expression of VP1.
  • the growth curve is shown in Figure 1.
  • the mice inoculated with CMS5-VP1/F5 cells started to regress on the 8th day after inoculation, and the tumors completely regressed after the 10th day.
  • the tumors in mice inoculated with 1 ⁇ 10 6 CMS5-VP1/B5G2 cells grew slowly, and the tumors completely regressed on the 16th day after inoculation, while the tumors in mice inoculated with 1 ⁇ 10 6 CMS5-VP1/B5G2 cells started on the 14th day.
  • the tumor tissue was taken, minced, digested with trypsin for half an hour, washed three times with RPMI160 complete medium, and the tumor cells were collected. 1 ⁇ 10 7 tumor cells were taken and lysed in 1 mL of cell lysate at 4°C for 2 hours, and the cell supernatant was collected by centrifugation at 13,000 rpm, and frozen at -20°C.
  • the tumor growth trend of the tumor-bearing mice inoculated with 3 ⁇ 10 6 CMS5-VP1/E2 was not significantly different from that of the control group CMS5.
  • the tumor grew rapidly on the 18th day, and the tumor volume was >2000mm3 on the 20th day, while the inoculated 1 ⁇ 10 6 CMS5
  • the tumor-bearing mice of -VP1/E2 grew relatively slowly, and the tumor size was 1000 on the 20th day after inoculation.
  • the tumor-bearing mice were euthanized, and some tumor tissues were digested with trypsin and the cells were collected.
  • 1 ⁇ 10 7 tumor cells were lysed with 1 mL of cell lysate at 4°C for 2 hours, and the cell supernatant was collected by centrifugation at 13,000 rpm, and frozen at -20°C.
  • CMS5-VP1 cells In order to further determine the stability of CMS5-VP1 cells, the lysates of CMS5-VP1/B5G2 and CMS5-VP1/E2 cells were taken for western blot analysis. The results showed that VP1 protein was expressed in the tumor tissue of CMS5-VP1 tumor-bearing mice. .
  • the plasmid pcDNA3.1-VP2 was synthesized by Nanjing GenScript, inserted into the multi-cloning site of vector pcDNA3.1 by endonuclease Kpn I/Xho I, and the correctness of the insertion site was confirmed by sequencing.
  • the pVAX1-VP1/DH5 ⁇ and pcDNA3.1-VP2/DH5 ⁇ glycerol bacteria were streaked on LB plates (containing corresponding antibiotics such as Amp or Kan), and cultured in a 37°C incubator overnight.
  • the pVAX1-VP1/DH5 ⁇ and pcDNA3.1-VP2/DH5 ⁇ monoclonal colonies were respectively picked and placed in 5 mL of LB liquid medium (containing corresponding antibiotics such as Amp or Kan), and incubated at 37°C with shaking on a shaker overnight.
  • the plasmids of pVAX1-VP1/DH5 ⁇ and pcDNA3.1-VP2/DH5 ⁇ cultured overnight were extracted with plasmid mini-suction kit.
  • the endonuclease Hind III/Xho I was subjected to double digestion to obtain the linearized vector pcDNA3.1 and the target gene VP1, respectively.
  • the digestion system is as follows in Table 5, The digestion reaction was carried out at 37°C for 4h.
  • the enzyme ligation system is determined to carry out the enzyme ligation reaction, and the reaction system is shown in Table 6 below.
  • the enzyme ligation product was added to 50 ⁇ L of DH5 ⁇ competent cells, ice-bathed for 30 min, followed by a water bath at 42 °C for 90 s, 500 ⁇ L of LB liquid medium was added to the cells, and incubated at 37 °C with a shaker at 100 rpm for 30 min. After the end, centrifuge at 13,000 rpm, discard the supernatant, resuspend the bacterial cells with 200 ⁇ L of LB liquid medium, spread on LB plates (containing Amp), and cultivate overnight in a 37°C incubator.
  • the plasmids were extracted with the QIAGEN mid-suction plasmid kit, and the plasmid concentrations were determined by UV260/280. The plasmid concentrations are shown in Table 7 below.
  • the plasmid was identified by Hind III/Xho I double-enzyme digestion, and the identification results showed that the size of the band obtained by double-enzyme digestion was correct, indicating that the plasmid was correct.
  • the 293FT cells were recovered. After the cells were stably cultured, the cells were digested with trypsin, centrifuged at 1000 ⁇ g for 5 min to collect the cells, resuspended in 1 mL of DMEM complete medium, and 1 ⁇ L of the cell suspension was diluted 100 times and stained with trypan blue. count the number of cells;
  • the cell dilution solution was added to a 6-well plate, 2 mL per well, and cultured overnight at 37°C in a 5% CO2 incubator;
  • the pcDNA3.1-VP1, pcDNA3.1-VP2, pcDNA3.1-EGFP plasmids were mixed according to the ratio in Table 8, and co-transfected with reference to the Lipofectamine 2000 operation manual;
  • the cell lysate was centrifuged at 5000 ⁇ g for 10 min, and the supernatant was collected, which was the pseudovirus solution; each group of pseudoviruses was diluted with DMEM complete medium, the initial dilution gradient was 1:1000, and the 10-fold ratio was serially diluted to 7 gradients;
  • Each ladder pseudovirus dilution was added to a 96-well plate of 293FT cells cultured overnight, 100 ⁇ L of pseudovirus dilution per well, 2 duplicate wells per gradient, and cultured in a CO2 incubator at 37°C for 72h.
  • the prepared pseudovirus did not have the ability to infect 293FT cells.
  • the fluorescent protein expression intensity was high.
  • 20 ⁇ L of the pseudovirus suspension in each group was taken, and the anti-VP1 mouse serum was used as the primary antibody for immunoblotting detection.
  • the cell lysate was centrifuged at 5000 ⁇ g for 10 min, and the supernatant was collected, which was the pseudovirus suspension; each group of pseudoviruses was diluted with DMEM complete medium, the initial dilution gradient was 1:1000, and 7 gradients were serially diluted at a 10-fold ratio. ;
  • Each ladder pseudovirus dilution was added to a 96-well plate of 293FT cells cultured overnight, 100 ⁇ L of pseudovirus dilution per well, 2 duplicate wells per gradient, and cultured in a CO2 incubator at 37°C for 72h.
  • the pseudovirus packaged in 1 ⁇ 10 7 cells with fluorescent protein particle Zs-Green as a reporter gene did not have the ability to infect 293FT.
  • pE2-GFP and pRwB were used as reporter genes to package pseudovirus in 1 ⁇ 10 7 cells.
  • the pseudovirus was packaged and matured, and the pseudovirus suspension was taken.
  • the initial dilution was 1:1000, and the 10-fold serial dilution was carried out in 7 gradients.
  • the experiment was carried out to infect 293FT cells in a 96-well plate, and the fluorescence in each well was observed. strength.
  • the ability of pseudovirus with RwB as reporter gene to infect 293FT was weak.
  • Example 1 A DNA vaccine against Merkel cell carcinoma
  • the nucleotide sequence shown in SEQ ID NO.1 was cloned into the pVAX1 vector, and the enzyme cleavage sites used for the cloning were XbaI and HindIII.
  • the specific steps of cloning are conventional methods in the art, which will not be repeated here.
  • the cloning method includes PCR amplification, restriction enzyme digestion, ligation and related steps.
  • pVAX1-MCV-VP1 The recombinant vector obtained by successfully cloning the nucleotide sequence shown in SEQ ID NO.1 into the pVAX1 vector is named pVAX1-MCV-VP1 according to the conventional naming convention in the art.
  • the identification method is a conventional method in the field, and will not be repeated here.
  • double-enzyme digestion identification or sequencing identification After double digestion with XbaI and HindIII, a DNA band of about 1.3kb could be observed.
  • pVAX1-MCV-VP1 was amplified using conventional kits in the field, such as the endotoxin-free plasmid extraction kit (catalog number DP117) of Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • Example 1 The only difference between this example and Example 1 is that the nucleotide sequence shown in SEQ ID NO.1 is replaced by the nucleotide sequence shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 .
  • the recombinant vector obtained by successfully cloning the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 into the pVAX vector is named pVAX-MCV according to the conventional naming convention in the art - VP1-1, pVAX-MCV-VP1-2, pVAX-MCV-VP1-3.
  • Example 3 A protein vaccine against Merkel cell carcinoma
  • Cloning connect the nucleotide sequence shown in SEQ ID NO.3 with a 6 ⁇ His tag at its C-terminus, and clone it into the pET28a vector through endonuclease XhoI and NcoI to obtain pET28a- VP1/Rosetta cells (by ) were stored in a -80°C freezer.
  • the enzyme cleavage sites used for cloning were NcoI and XhoI. The specific steps of cloning are not repeated here.
  • the cloning method includes PCR amplification, enzyme digestion, ligation and related steps.
  • the recombinant vector obtained by successfully cloning the nucleotide sequence shown in SEQ ID NO.3 into the pET28a vector is named pET28a-VP1 according to the conventional naming convention in the art.
  • Protein expression any expression system capable of realizing prokaryotic protein expression can be used for expression.
  • expression in BL21 or Rosetta cells the specific expression steps are carried out with reference to the user manual of BL21 or Rosetta cells, which will not be repeated here.
  • the specific purification method refers to the product instructions of purification system and nickel column.
  • the first step of dialysis conditions 4 °C, change the dialysate every 6 hours, each 2L.
  • the external dialysis fluids are: PBS+0.5M NaCl, PBS+0.4M NaCl, PBS+0.3M NaCl, PBS+0.2M NaCl, PBS+0.1M NaCl, PBS; the purpose of this step of dialysis is to remove the effluent in the eluate. Excess salt component; after dialysis with PBS, the desalted eluent containing the target protein is obtained;
  • a conventional concentration method eg, PEG8000 powder concentration
  • MCV-VP1 can self-assemble into VLPs in eukaryotic systems, usually the envelope protein VP2 of MCV is also involved in this process, but it has not been reported that VP1 can also self-assemble into VLPs in prokaryotic systems, especially in the absence of VP2 with participation. Since the assembly of VLPs may be largely affected by external conditions, mainly including factors such as pH, strength of sodium and calcium ions, etc., different external conditions were adopted in the process of exploring the in vitro assembly of VLPs in prokaryotic systems.
  • MCV-VP1 with the nucleotide sequence shown in SEQ ID NO. 3 could be expressed as a soluble protein in E. coli and self-assembled to form particles of different sizes in vitro.
  • Transmission electron microscopy experiment The recombinant protein obtained after the final dialysis (ie, protein vaccine VP1) was centrifuged through a 30%-60% sucrose density gradient; then samples between different component layers were drawn to verify the position of the target protein after centrifugation .
  • the target protein obtained by sucrose density gradient centrifugation was stained with phosphotungstic acid staining solution, and the presence of virus-like particles was observed by transmission electron microscopy under the condition of a voltage of 80kV.
  • SDS-PAGE The gel concentration is 12%, and the electrophoresis conditions are as follows: the stacking gel is 80V for about 30 minutes, and the separating gel is 120V for about 1 hour.
  • Example 4 A protein vaccine against Merkel cell carcinoma
  • Example 3 The only difference between this example and Example 3 is that the nucleotide sequence shown in SEQ ID NO.3 is replaced by the nucleotide sequence shown in SEQ ID NO.1.
  • Streak culture of glycerol bacteria Take out the pET28a-VP1/Rosetta glycerol bacteria with His tag prepared in Example 3 from a -80°C refrigerator, and thaw at room temperature. In the ultra-clean bench, dip the glycerol bacterial solution with an inoculating needle and streak it on the antibiotic LB plate containing 100 ⁇ g/mL kanamycin sulfate (Kan), and culture in a constant temperature incubator at 37 °C overnight.
  • Kan kanamycin sulfate
  • a feed medium with a total volume of 2.5 L was prepared, placed in a feed bottle, together with each feed tube and feed head, and sterilized at 115° C. for 20 minutes.
  • a sterile refill bottle is filled with ammonia water for use.
  • pH control automatically add ammonia water and control it at about pH 7.0;
  • DO control greater than or equal to 40%, set 40%, below this value, increase the speed and gas control
  • Control of rotation speed adjust according to dissolved oxygen to meet the demand of dissolved oxygen, up to 600 rpm;
  • Foam control intermittently add defoamer (the principle of supplementation is to add as little defoamer as possible, and control the liquid level without spraying the tank).
  • Process detection measure the OD600 value every hour and record the fermentation situation.
  • the tank was closed, and the fermentation broth was centrifuged at 8000 rpm for 30 minutes. The supernatant was discarded, the precipitate was washed twice with sodium phosphate (pH 7.0) buffer, and collected for later use.
  • the MCV vaccine antigen VP1 used in this study was expressed in E. coli prokaryotic expression system and induced by IPTG.
  • the codon-optimized VP1 was constructed into the prokaryotic expression vector pET28a by molecular cloning technology, and the plasmid was named pET28a-VP1.
  • the correctness of the plasmid was confirmed by sequencing and enzyme digestion.
  • the pET28a-VP1 plasmid with the correct sequence was chemically transformed into E.
  • coli expression strain Rosetta competent cells named pET28a-VP1/Rosetta, and the expression strain was inoculated at 1:100 in LB liquid medium containing 100 ⁇ g/mL Kan, 37 After shaking and culturing overnight at °C, the bacterial liquid cultured overnight was divided into 1.5 mL sterile centrifuge tubes, and 750 ⁇ L of bacterial liquid in each tube was mixed with 250 ⁇ L of 60% sterile glycerol.
  • the OD 600 was 24.9 after inoculation in a fermenter at 37°C for 10.5h, the temperature was lowered to 30°C, VP1 protein expression was induced with 0.5mM IPTG for 3h, the OD 600 reached 35.2, and the fermentation was collected in the tank. After centrifugation at 8000g for 30 minutes, the cells were collected and weighed to obtain 400g of wet bacteria.
  • 0.1 g of bacteria were resuspended in 1.5 mL of sterilization buffer, and centrifuged after sterilization by a high-pressure homogenizer. The sterilized supernatant and precipitate were collected respectively, and the precipitate was resuspended in 1.5 mL of lysis buffer and lysed overnight. After the lysate was centrifuged, the supernatant and the pellet were collected respectively.
  • the results of SDS-PAGE electrophoresis showed that the size of the target protein VP1 was 46kDa, and lanes 2 and 3 had obvious bands around 45kDa. From the results of SDS-PAGE, it could be determined that the VP1 induced and expressed in the fermenter at 30°C mainly formed inclusion bodies.
  • the immunoblotting results using anti-His tag mouse polyclonal antibody as the primary antibody further confirmed that the target protein VP1 mainly formed inclusion bodies.
  • the VP1 recombinant protein was purified by nickel ion affinity chromatography.
  • Example 5 Take 10 g of the MCV-VP1 expressing cells prepared in Example 5, and resuspend the cells with 150 mL of sterilization buffer (0.1 M NaH 2 PO 4 , 20 mM Tris, 0.3 M NaCl, pH 7.0). Homogenize with a high pressure homogenizer for 30 minutes, centrifuge at 8000 ⁇ g for 30 minutes, and collect the cells.
  • sterilization buffer 0.1 M NaH 2 PO 4 , 20 mM Tris, 0.3 M NaCl, pH 7.0.
  • the cells were resuspended in 150 mL of lysis buffer (0.1M NaH 2 PO 4 , 20 mM Tris, 0.3 M NaCl, 8 M urea, pH 7.0), adjusted to pH 7.0, and lysed at 4°C overnight.
  • lysis buffer 0.1M NaH 2 PO 4 , 20 mM Tris, 0.3 M NaCl, 8 M urea, pH 7.0
  • Ni-NTA was equilibrated with 5 column volumes of equilibration buffer (0.1 M NaH 2 PO 4 , 20 mM Tris, 0.3 M NaCl, 8 M urea, pH 7.0).
  • wash buffer 0.1M NaH2PO4 , 20mM Tris, 0.3M NaCl, 8M urea, 20mM Imidazole, pH 7.0
  • Elute the target protein with eluent (0.1M NaH 2 PO 4 , 20mM Tris, 0.3M NaCl, 8M urea, 500mMImidazole, pH7.0) at a flow rate of 2mL/min, and collect the eluted target protein in steps .
  • the eluted target protein was detected by SDS-PAGE and immunoblotting.
  • the VP1 expressed by the inclusion bodies was resuspended in the sterilization buffer, and then mechanically sterilized by a high-pressure homogenizer.
  • the sterilized pellet was lysed overnight with a lysate containing 8M urea, and the supernatant obtained by centrifugation was placed on a GE AKATA prime machine.
  • VP1 was purified by nickel ion affinity chromatography.
  • the purified protein was detected by SDS-PAGE, and the results are shown in Figure 3, where M represents the protein prestained Marker (Tiangen Biochemical Technology, MP206); 1 is the supernatant of bacteria breaking; 2 is the supernatant of lysis; 3 is the flow-through 4 is a 20 mM imidazole wash; 5 is a 100 mM wash; 6 is a 250 mM eluent; 7 is a 500 mM wash; 8 is a 0.5 M sodium hydroxide wash. It can be seen from Figure 3 that the target protein eluted from 250 mM and 500 mM imidazole is a single band.
  • M represents the protein prestained Marker (Tiangen Biochemical Technology, MP206)
  • 1 is the supernatant of bacteria breaking
  • 2 is the supernatant of lysis
  • 3 is the flow-through 4 is a 20 mM imidazole wash
  • 5 is a 100 mM wash
  • 6 is a 250
  • the urea was gradually removed by dialysis bag dialysis. After overnight dialysis in a buffer containing 2M urea, dialysis was performed in different dialysis buffers overnight to completely remove urea to renature VP1. The renatured VP1 spontaneously assembles into protein particles with pentamers as subunits.
  • Buffer1 100mM NaH2PO4, 20mM Tris, pH 7.2.
  • Buffer2 100mM NaH2PO4, 20mM Tris, 2M(NH4)2SO4, 2mM CaCl2, pH7.2.
  • Buffer3 100mM NaH2PO4, 20mM Tris, 1M NaCl, pH 7.2.
  • Buffer4 100mM NaH2PO4, 20mM Tris, 150mM NaCl, 2mM CaCl2, pH7.2.
  • Buffer5 20mM MOPS, 0.3M NaCl, pH 7.0.
  • Embodiment 7 MCV-VP1 reassembly process research
  • Covalent disulfide bonds play an important role in the formation of higher-order structures of proteins.
  • Reducing reagents such as DTT and high concentration of urea can respectively break the wrong covalent disulfide bonds and ionic bonds to open the higher-order structure of the protein and make the protein exist in the form of monomer molecules.
  • This process is protein denaturation.
  • concentration of reducing agents such as DTT and urea
  • the VP1 purified under denaturing conditions was gradually removed by dialysis to renature the urea in the protein solution. The specific method is as follows:
  • Buffer1 100mM NaH2PO4, 20mM Tris, pH 7.2.
  • Buffer2 100mM NaH2PO4, 20mM Tris, 2M(NH4)2SO4, 2mM CaCl2, pH7.2.
  • Buffer3 100mM NaH2PO4, 20mM Tris, 1M NaCl, pH 7.2.
  • Buffer4 100mM NaH2PO4, 20mM Tris, 150mM NaCl, 2mM CaCl2, pH7.2.
  • Buffer5 20mM MOPS, 0.3M NaCl, pH 7.0.
  • rVP1 The reassembled (also known as self-assembled) recombinant VP1 was named (rVP1), and then rVP1 was filtered and sterilized with a 0.22um syringe filter, and the specific structure of rVP1 was detected by Nu-PAGE. The particle size of rVP1 was measured by caliper.
  • Wild-type MCV is a 72-hedral virus particle composed of VP1 pentamer as the basic unit. It has been reported in the literature that the recombinantly expressed polyoma virus capsid protein VP1 also exists in a multimer structure. Nu-PAGE was used to detect whether the VP1 structural protein formed under different assembly processes is a pentamer as the basic unit. The results of Nu-PAGE electrophoresis are shown in Figure 4. 1-5 in Figure 4 represent the assembly of VP1 and M under the above different reassembly conditions, respectively, and the protein is not pre-stained with Marker. The results show that the assembly of VP1 under different assembly conditions ( rVP1) exists in the form of multimers in addition to forming pentamers.
  • the size of the self-assembled VP1 (rVP1) particles was detected by Malvern nanoparticle size analyzer, and the results showed that the self-assembled VP1 (rVP1) structural protein formed particles of different sizes.
  • the disassembly-reassembly process of the self-assembled VP1 structural protein was studied with DTT and EGTA, hoping to obtain recombinant protein particles closer to the size of wild-type MCV particles through the reassembly process.
  • the self-assembled VP1 (rVP1) structural protein was disassembled with 10 mM DTT and 10 mM EGTA, and then the VP1 reassembly process was explored under different reassembly conditions, and the redox agent (GSSG- GSH) conditions for the reassembly process.
  • VP1 After VP1 disassembly, it was dialyzed against reassembly buffer (50 mM Tris, 0.8 M (NH 4 ) 2 SO 4 , 0.2 M NaCl, 0.5 mM GSH, 4.5 mM GSSG, 2 mM CaCl 2 , pH 6.4) for 48 hours, followed by The protein solution was removed by dialysis overnight against buffer (50 mM Tris, 0.2 M NaCl, pH 7.0) (NH 4 ) 2 SO 4 and CaCl 2 . Reassembled VP1 (rVP1) was obtained by these processes.
  • reassembly buffer 50 mM Tris, 0.8 M (NH 4 ) 2 SO 4 , 0.2 M NaCl, 0.5 mM GSH, 4.5 mM GSSG, 2 mM CaCl 2 , pH 6.4
  • the reassembled VP1 (rVP1) was detected by SDS-PAGE and immunoblotting, and the pentameric structure of VP1 was detected by non-reducing electrophoresis.
  • the rVP1 showed a unique band under denaturation conditions. Detected by Nu-PAGE of Themofisher Company, the molecular weight of VP1 monomer is 46kDa, and the molecular weight of pentamer is about 250kDa. Therefore, it can be considered that the band at 250kDa is the VP1 pentamer structure m. From this result, it is believed that the rVP1 obtained by the reassembly process mainly uses pentamer as the basic unit to form protein particles.
  • rVP1 and low temperature-induced soluble VP1 were measured by a nanoparticle particle size analyzer. Compared with VP1 self-assembled particles, rVP1 particles were more uniform, with a particle size of 115.3 nm. The particles of sVP1 were much larger than those of rVP1, which indicated that the presence of redox-reductants during the reassembly process contributed to the stability of the structure during the reassembly process.
  • the mouse-specific MCV-VP1 antibody titer and the footpad thickness after DTH were detected 7 days after the final immunization (B/C/ D).
  • the results showed that anti-MCV-VP1-specific antibody titers could be detected in the protein vaccine immunization groups of different doses, but the highest antibody titer was observed in the 10 ⁇ g group (B in Figure 5).
  • the DTH level of the 3.3 ⁇ g group was the highest, the DTH level of the 10 ⁇ g group was also relatively high (C/D in FIG. 5 ). Therefore, considering the results of specific antibody levels and DTH, 10 ⁇ g was finally selected as the optimal dose of protein vaccine, and this dose was used in the following experiments (VP1 in the figure refers to the protein vaccine immunization group).
  • MHC-I/MHC-II epitope peptide (MCV-VP1peptide) was predicted and synthesized, and the Splenocytes isolated from mice.
  • MMV-VP1peptide protein vaccine immunization group
  • the results showed that the levels of IFN- ⁇ and TNF- ⁇ in CD8 + T cells in the protein vaccine immunization group (MCV-VP1) were significantly higher than those in the PBS immunized group (Vehicle), while the levels of IL-4 and CD8 + T cells in CD4 + T cells were significantly higher than those in the PBS immunized group (Vehicle).
  • the level of Granzyme B also showed the same trend (E in Figure 5).
  • mice used in this experimental example were 6-8 week old BALB/c and C57BL/6 mice, which were purchased from JST.
  • the DNA vaccine was immunized by intramuscular injection combined with electric pulse
  • the protein vaccine was immunized by intramuscular injection of protein and aluminum adjuvant.
  • DTH Delayed type hypersensitivity
  • Enzyme-linked immunosorbent assay The blood samples of mice were collected on the 21st day, and the specific antibody titers of serum were determined by ELISA method. When semi-quantitative detection of antibody concentration in mouse serum is required, a standard curve is added on the basis of conventional ELISA, and the concentration of antibody in mouse serum is determined according to the concentration of the standard curve.
  • BALB/c who had completed the immunization program were collected blood from the fundus on the 28th day after the primary immunization, and left at 4°C overnight. After centrifugation at 6000rpm for 10 minutes, serum was collected. The level of anti-VP1 antibody in serum was detected by ELISA.
  • the specific method is as follows:
  • rVP1 was diluted to 2 ⁇ g/mL with antigen coating solution, and 96-well ELISA plate was coated with 100 ⁇ L/well of antigen dilution solution, overnight at 4°C.
  • HRP-labeled goat anti-mouse IgG and HRP-labeled goat anti-rabbit IgG were diluted 1000-fold with 1 ⁇ PBST containing 2% nonfat dry milk, respectively. After the primary antibody incubation, the primary antibody was shaken off, washed 5 times with 1 ⁇ PBST, and dried. Add diluted HRP-labeled goat anti-mouse IgG to 100 ⁇ L/well, and add HRP-labeled goat anti-rabbit IgG to the positive control. Incubate for 1 hour at 37°C.
  • Reading Read the light absorption value with a microplate reader at a wavelength of 450nm/620nm.
  • Splenocyte-specific stimulation performed in a sterile environment, the mice 7 days after the final immunization were euthanized by necking, the spleen was removed, and ground into a single-cell suspension; cells were harvested by centrifugation, resuspended in red blood cell lysate, and terminated by lysing in PBS containing FBS Lyse; filter, wash with PBS and resuspend cells in 1640 complete medium containing anti-CD28 (1:5000 dilution); count the prepared single cell suspension and plate with 1 ⁇ 10 6 cells/well; each cell Three replicates were set for mouse samples, including unstimulated group, VP1-specific peptide stimulation group (MCV-VP1 Peptide), and phorbol ester/ionomycin positive stimulation group (PMA+Ino).
  • MMV-VP1 Peptide VP1-specific peptide stimulation group
  • PMA+Ino phorbol ester/ionomycin positive stimulation group
  • the working concentration of polypeptide is 10 ⁇ g/mL
  • the working concentration of PMA is 0.1 ⁇ g/mL
  • the working concentration of Ino is 1 ⁇ g/mL.
  • the mixture should contain BFA (Greeneldin A) in a ratio of 1:1000 to inhibit the secretion of cytokines to the outside of the cells; cultured at 37° C., 5% CO 2 for 6 hrs; collected stimulated cells by centrifugation. Flow cytometry detection.
  • Flow cytometry dead and alive staining: using EF780-APC-Cy7 antibody, darkened on a shaker at room temperature; cell surface staining: using surface antibody staining solution (CD3-FITC/CD4-BV421/CD8-Percp-Cy5.5), The staining procedure was the same as that of dead and alive cells; intracellular staining: Foxp3/transcription factor staining kit (eBioscience) was used; flow cytometry LSRFortssa (Becton) was used for detection.
  • Protein vaccines mainly activate the body's humoral immune response, while the cellular immune response is weak. Therefore, the DNA vaccine against Merkel cell carcinoma prepared in Example 1 was further verified.
  • the pVAX1-MCV-VP1 (that is, DNA vaccine) obtained in Example 1 was immunized three times by intramuscular injection with a fixed dose of DNA vaccine combined with electric pulses at different time intervals. Mice were able to obtain the highest DTH and specific antibody responses (A, B in Figure 6).
  • DNA vaccines with different doses ranging from 12.5 ⁇ g/mice to 100 ⁇ g/mice were selected to immunize mice with a strategy of intramuscular immunization with an interval of one week and a total of three immunizations. The optimal immunization dose was determined to be 100 ⁇ g.
  • mice immunized with the pVAX1-MCV-VP1 DNA vaccine were stimulated with a specific peptide pool, and the levels of IFN- ⁇ in CD4 + T cells and TNF- ⁇ in CD4 + T cells were significantly increased, while the More significant is the IFN- ⁇ of CD8 + T cells, which is about 5 times higher (E, F in Fig. 6 ).
  • mice were intramuscularly immunized with pVAX1-OVA without electrical pulses;
  • mice were intramuscularly immunized with pVAX1-OVA without electrical pulses;
  • mice were intramuscularly immunized mice with pVAX1-OVA without electrical pulses;
  • mice were intramuscularly immunized mice with pVAX1-OVA without electrical pulses;
  • mice were intramuscularly immunized mice with pVAX1-OVA without electrical pulses;
  • mice were immunized mice with pVAX1-MCV-VP1 intramuscular injection combined with electrical pulses on days 0, 7, and 14;
  • pVAX1-MCV-VP1 2-3 were used on days 0, 14, 28
  • the mice were immunized with pVAX1-MCV-VP1 intramuscular injection combined with electric pulse on day 0;
  • pVAX1-MCV-VP1 n 2-3 were immunized with pVAX1-MCV-
  • this example verifies that the DNA vaccine prepared in Example 1 and Whether the combination of the protein vaccine prepared in Example 3 can induce a higher level of cellular and humoral immune responses at the same time.
  • mice were immunized on days 0, 14 and 28, regardless of whether it was a DNA vaccine or a protein vaccine, the corresponding optimal immunization doses explored in Example 8 and Example 9 were used.
  • D represents a dose of DNA vaccine
  • P represents a dose of protein vaccine
  • the immunization strategy of one dose of DNA vaccine plus two doses of protein vaccine could induce the highest level of DTH response, followed by D-D-P, D-D-D and P-P-P.
  • the optimal immunization strategy was D-P-P, followed by P-P-P, D-D-P, and D-D-D (B in Figure 7). From the comparison between different immunization strategies, the immunization strategy of one dose of DNA vaccine plus two doses of protein vaccine (DPP) can achieve higher levels of anti-VP1 antibody and DTH response, which indicates that the immunization strategy can induce both humoral and cellular immunity reaction.
  • Cytokine rhGM-CSF was purchased from North China Pharmaceutical Jintan Biopharmaceutical Co., Ltd.
  • Recombinant murine IFN- ⁇ 2b was purchased from Beijing Yiqiao Shenzhou (50525-MNAY).
  • CpG1826 was synthesized by Shanghai Jereh Biotechnology.
  • MCV therapeutic vaccine was prepared with rhGM-CSF and IFN- ⁇ 2b (rmIFN- ⁇ 2b) or CpG, R848 or MPL combined with Al(OH) 3 as adjuvant, and the antitumor effect of MCV therapeutic vaccine was studied.
  • R848 was purchased from Invivogen (tlrl-r848).
  • the fluorescently labeled antibody Biolegend or BD company used in the flow cytometry detection in the experiment.
  • Puromycin was purchased from Shanghai Yisheng Biotechnology Co., Ltd.
  • RPMI1640 fetal bovine serum (FBS), trypsin and penicillin/streptomycin double antibody for cell culture were purchased from BI Company.
  • Hemocytometer trypan blue, purchased from Thermo Fisher.
  • mice used in the experiment were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed in the SPF clean room of the Laboratory Animal Science Department of Fudan University. Under the guidance of animal protection guidelines.
  • the lentiviral plasmid pcDH-VP1 was transfected into the tumor cell CMS5 in the BALB/c background by lipofection method, and the cell line CMS5-VP1/ E2.
  • the tumorigenic experiment carried out in BALB/c mice confirmed that the tumor model was constructed by subcutaneous injection of 1 ⁇ 10 6 cells. (see basic example 1)
  • CMS5-VP1/E2 cells cryopreserved at -80°C were recovered.
  • the specific method is as follows: after the CMS5-VP1/E2 cell cryopreservation tube is thawed in a 37°C water bath, washed twice with RPMI1640 complete medium, and centrifuged at 1500 rpm for 5 minutes to collect the cells. The cells were resuspended in RPMI1640 complete medium containing 5 ⁇ g/mL Puromycin, and cultured overnight at 37°C in a 5% CO 2 incubator in a 10 cm cell culture dish.
  • the cells were washed twice with PBS and centrifuged at 1500 rpm for 5 minutes to collect the cells. After the cells were resuspended in PBS, the cells were filtered through a 40- ⁇ m mesh screen to prepare a single-cell suspension. Some cells were diluted, stained with trypan blue, and counted with a cell counting plate under a microscope. The single cell suspension was diluted to 1 ⁇ 10 7 /mL with PBS, and placed in a 4°C ice box for later use.
  • mice BALB/c female mice aged 6-8 weeks were randomly divided into cages, 5 mice in each group.
  • the CMS5-VP1 cell suspension was subcutaneously inoculated on the back of the mice, and the inoculation volume was 1 ⁇ 10 6 /100 ⁇ L.
  • MCV therapeutic vaccines were injected intramuscularly into the hindlimbs of mice, respectively.
  • the MCV therapeutic vaccine group and the other groups were injected intramuscularly 5, 12, and 19 days after tumor inoculation, and each group was:
  • a tumor cell line CMS5-VP1 stably expressing VP1 was constructed to establish a tumor model to evaluate the inhibitory effect of MCV therapeutic vaccine on tumor growth.
  • the volume of the tumor was calculated according to the formula.
  • Tumor growth curves were made with GraphPad Prism, as shown in Figure 8. Statistical analysis showed that there was a certain difference in the tumor growth curve between the Al(OH) 3 control group and the PBS placebo group.
  • the tumor growth curves of each MCV therapeutic vaccine group were significantly different from those of the Vac vaccine group and the Al(OH) 3 control group.
  • the tumor growth curve of the three groups was significantly different from that of the Al(OH) 3 control group, and also had a certain inhibitory effect, but was not significantly different from the Vac vaccine group.
  • MCV therapeutic vaccine was prepared with R848, CpG, etc. combined with Al(OH) 3 as adjuvant, and the anti-tumor effect of MCV therapeutic vaccine was studied.
  • CpG1826 was synthesized by Shanghai Jierui Biotechnology; R848 was purchased from invivogen (tlrl-r848); aluminum hydroxide [Al(OH) 3 ] was purchased from Brenntag.
  • Antibodies related to flow detection were purchased from Biolegend or ebioscience.
  • mice 6-8-week-old female BALB/c mice used in the experiment were purchased from Shanghai Sipple-Bikai Laboratory Animal Co., Ltd. and kept in Shanghai Sipple-Bike Laboratory Animal Co., Ltd. SPF clean All animal experiments were carried out under the guidance of animal protection guidelines.
  • the lentiviral plasmid pcDH-VP1 was transfected into the BALB/c background tumor cells CMS5 by lipofection, and the cell lines stably expressing VP1 were screened under the pressure of 5 ⁇ g/mL Puromycin. CMS5-VP1/E2.
  • BALB/c female mice aged 6-8 weeks were randomly divided into cages, 6 mice in each group.
  • the MCV tumor model was established by subcutaneously inoculating 1 ⁇ 10 6 /100 ⁇ L of CMS5-VP1 cell suspension into the back of mice. (see basic example 1)
  • the recombinant protein rVP1 was diluted with buffer, mixed with aluminum adjuvant Al(OH) 3 at a ratio of 1:1, and shaken at low speed in a shaker at 4°C overnight (to facilitate the uniform adsorption of antigen on Al(OH) 3 adjuvant). dose), as a semi-finished product of MCV recombinant protein vaccine.
  • the final vaccine per unit was 10 ⁇ g rVP1, 500 ⁇ g Al(OH) 3 , 10 ⁇ g CpG, and 10 ⁇ g R848.
  • mice are divided into 6 groups, including:
  • CpG/R848/Al CpG 10 ⁇ g; R848 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • mice On the 33rd day after tumor inoculation, the mice were euthanized, and the spleen was removed to prepare a single-cell suspension. After in vitro stimulation with VP1 antigen, the level of antigen-specific cellular immune response was detected. Statistical analysis was performed with GraphPad prism software.
  • Figure 9 shows the tumor-bearing pictures of mice in each vaccine group on the 33rd day after tumor inoculation.
  • VP1/CpG/Al group and VP1/R848/Al group vaccine can significantly delay the growth of CMS5-VP1 tumor-bearing mice, while VP1/CpG/R848/Al group compared with PBS untreated group, The difference in growth curve was the most significant.
  • the tumors of the mice were basically controlled, and the tumor size basically did not grow and had a tendency to be cleared.
  • the experimental results show that CpG and R848 adjuvant, whether used alone or in combination, can improve the therapeutic level of VP1/Al vaccine on MCV tumors, and the combination of the two has the most significant anti-tumor effect.
  • mice 33 days after tumor inoculation, the antigen-specific cellular immune responses induced by MCV vaccine in the spleen of mice were detected by flow cytometry.
  • the VP1/CpG/R848/Al vaccine group induced IL-2 and TNF- ⁇ secreted by CD4+ T cells.
  • the levels of , IFN- ⁇ and Granzyme B, IL-2, TNF- ⁇ and IFN- ⁇ induced by CD8+ T cells were significantly higher than other vaccine groups, that is to say, VP1/CpG/R848/Al vaccine group
  • T cells can kill tumor cells by secreting effectors such as IL-2, TNF- ⁇ , IFN- ⁇ , and Granzyme B.
  • Preventive vaccines are usually given before pathogen infection occurs.
  • the vaccine induces the host immune system to produce antigen-specific antibodies and immune memory.
  • the immune system can effectively block the invasion or expansion of pathogens.
  • the prepared MCV therapeutic vaccine could significantly inhibit tumor growth. Therefore, whether the MCV therapeutic vaccine can achieve the killing effect on tumors by enhancing the T cell immune response is tested by animal experiments.
  • the immunization procedure was as follows: injection on day 0 and day 14, and blood collection on day 28.
  • the MCV therapeutic vaccine was injected with adjuvant GM-CSF+IFN- ⁇ , TLR9 agonist CpG1826, TLR7/8 agonist R848 and TLR4 agonist MPL as adjuvant.
  • Therapeutic vaccines Vac/G+I, Vac/C, Vac/R, Vac/M and MCV recombinant protein vaccine Vac and Al(OH) 3 adjuvant control group were immunized twice (14 days), and the conditions of each group were as follows:
  • Vac/G+I group 10 ⁇ g rVP1/10 ⁇ g GM-CSF/1 ⁇ g IFN- ⁇ /500 ⁇ g Al(OH) 3 ;
  • Vac/C group 10 ⁇ g rVP1/10 ⁇ g CpG/500 ⁇ g Al(OH) 3 ;
  • Vac/R group 10 ⁇ g rVP1/10 ⁇ g R848/500 ⁇ g Al(OH) 3 ;
  • Vac/M group 10 ⁇ g rVP1/25 ⁇ g MPL/500 ⁇ g Al(OH) 3 ;
  • Vac group 10 ⁇ g rVP1/500 ⁇ g Al(OH) 3 ;
  • Al(OH) 3 groups 500 ⁇ g Al(OH) 3 ;
  • VP1 group 10 ⁇ g rVP1;
  • PBS group 10 ⁇ L PBS.
  • Paw swelling 24 hours after injection of rVP1 or PBS is shown in Figure 12. As can be seen from the figure, the soles of the feet of the mice vaccinated with the MCV therapeutic vaccine were significantly swollen.
  • the strongest DTH response in each therapeutic vaccine group was the Vac/R group with R848 as adjuvant, followed by the Vac/C group with CpG as adjuvant and the Vac/M with MPL as adjuvant. group, they all showed extremely significant difference (****, p ⁇ 0.0001), and the difference of Vac/G+I with GM-CSF and IFN- ⁇ as adjuvant was more significant. This result is more consistent with the results of the development of MCV therapeutic vaccine to inhibit tumor growth.
  • DTH is a host immune response to pathogens or foreign proteins mediated by CD4 + or CD8 + T cells. Therefore, it is speculated that MCV therapeutic vaccine may achieve anti-tumor efficacy by inducing antigen-specific T cell immune responses.
  • 200ng/well sVP1 was used as antigen to coat a 96-well ELISA plate.
  • the serum of each sample was diluted with 1:800 as the initial dilution, and was serially diluted in 8 gradients.
  • the diluted serum was used as the primary antibody, 100 ⁇ L per well, and two duplicate wells for each dilution.
  • HRP-labeled goat anti-mouse was used as the secondary antibody.
  • the OD450/OD620nm reading light absorption value is 2.1 times of the OD value of the negative control group as the cutoff value.
  • the experimental group OD value > cut off is positive. If one of the two adjacent dilutions is positive and the other is negative, the upper One dilution is the antibody titer of the sample.
  • T cell depletion is a major problem in clinical treatment. Therefore, the need to consider when designing therapeutic vaccines is the ability to enhance the cellular immunity of the host. This has also become an important indicator for evaluating therapeutic vaccines in preclinical studies. Based on these assumptions, GM-CSF+IFN- ⁇ , CpG, R848 or MPL were selected as adjuvants for MCV therapeutic vaccines. Therefore, following the completion of serological tests, cellular immunological tests were carried out to determine the stimulating effect of these novel adjuvants on cellular responses.
  • mice splenocytes were collected 14 days after the secondary immunization, 1 ⁇ 10 6 splenocytes were stimulated in vitro with 2 ⁇ g of recombinant protein for 12 hours, and then blocked with 1 ⁇ BFA for 4 hours.
  • the expression levels of TNF- ⁇ , IFN- ⁇ , and IL-2 in T cells were detected by flow cytometry to verify whether the MCV therapeutic vaccine can achieve anti-tumor effects by enhancing the immune response of T cells.
  • the single cells of lymphocytes were framed, and the viable cell population negative for Fixable Viability Dye eFluor TM 780 was framed, and then CD3 + T cells were framed, and the expressions of TNF- ⁇ , IFN- ⁇ and IL-2 in CD4 + and CD8 + T cells were detected respectively. level.
  • mice were euthanized, splenocytes were harvested, and cytokines were detected by flow cytometry.
  • mice After the mice were euthanized, they were soaked in 75% ethanol for 10 minutes. The spleen was taken and immersed in RPMI1640 complete medium.
  • the spleen cells stimulated overnight were taken, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded.
  • Fluorescently labeled antibodies were diluted in 1 ⁇ PBS containing 2% FBS. After blocking, centrifuge at 1500 rpm for 5 minutes, and shake off the supernatant. Cells were resuspended with diluted antibody and stained at room temperature for 15 minutes in the dark.
  • Fixation After surface staining, 150 ⁇ L of 1 ⁇ PBS was added to stop the reaction. Centrifuge at 1500 rpm for 5 minutes and shake off the supernatant. Add 200 ⁇ L of paraformaldehyde and fix for 7 minutes at room temperature in the dark.
  • Intracellular staining Fluorescently labeled antibodies were diluted in 1 ⁇ PBS containing 2% FBS. After paraformaldehyde fixation, centrifuge at 1500 rpm for 5 minutes, and shake off the supernatant. Washed twice with 1 ⁇ PBS. Cells were resuspended with diluted antibody and stained at room temperature for 15 minutes in the dark.
  • Flow cytometry detection After staining, 150 ⁇ L of 1 ⁇ PBS was added to stop the reaction. Centrifuge at 1500 rpm for 5 minutes and shake off the supernatant. Resuspend cells in 150 ⁇ L of 1 ⁇ PBS. Cytokine expression levels were detected by flow cytometry.
  • Fig. 15 showed that, compared with the Vac, Al(OH) 3 or VP1 group, the MCV therapeutic vaccine group could induce T cells to secrete TNF- ⁇ , IFN- ⁇ and IL-2, and the Vac/R vaccine group TNF- ⁇ , IFN- ⁇ and IL-2 The levels of IFN- ⁇ and IL-2 were significantly higher than other vaccine groups. This is positively correlated with the antitumor effect of MCV therapeutic vaccine.
  • MCV therapeutic vaccines achieve anti-tumor effects by inducing antigen-specific T cell immune responses.
  • Tumors were found to be in a slow-growing phase after completion of three MCV therapeutic vaccinations (days 19-21 post-tumor inoculation) in a previous study.
  • mice After 6-8 week old BALB/c mice were inoculated with CMS5-VP1 tumor cells, the MCV therapeutic vaccines Vac/G+I, Vac/C, Vac/R, Vac/M, and Vac were intramuscularly injected, and each control group . T cell immune responses were detected on the 20th and 34th days after tumor inoculation after three vaccinations.
  • the CMS5-VP1/E2 cells were digested with trypsin and prepared into a single cell suspension. The cells were counted and the cell viability rate was 95%. Cells were diluted to 1 x 107 /mL.
  • the tumor volume of the Vac/R group was significantly different from that of the other vaccine groups on the 19th day after tumor inoculation.
  • Three mice in each group were taken. After euthanasia, the spleens of the mice were taken to prepare spleen cell suspensions. 1 ⁇ 10 6 spleen cells were stimulated with 2 ⁇ g rVP1 for 20 hours, and simultaneously treated with 1 ⁇ g/mL ionomycin (Ionmycin) + 0.1 ⁇ g/mL phorbol ester (PMA) stimulation served as a positive control.
  • Ionmycin ionomycin
  • PMA phorbol ester
  • TNF- ⁇ , IFN- ⁇ and IL-2 secreted by CD4 + and CD8 + T cells were respectively detected, as shown in FIG. 17 .
  • the TNF- ⁇ and IFN- ⁇ secreted by CD4 + and CD8 + T cells in the Vac/R vaccine group were higher than those in the other vaccine groups, although the TNF- ⁇ secreted by CD4 + T cells and the IFN- ⁇ secreted by CD8 + T cells were not statistically significant differences, which may not be evident between groups due to the small sample size.
  • the tumor volume of each vaccine group was significantly different, and the inhibitory effect of Vac/R vaccine on tumor was particularly obvious.
  • the mice in each vaccine group were euthanized, the spleen was taken to prepare spleen cell suspension. 1 ⁇ 10 6 splenocytes were stimulated with 2 ⁇ g rVP1 and simultaneously stimulated with 1 ⁇ g/mL Ionmycin + 0.1 ⁇ g/mL phorbol ester (PMA) as a positive control.
  • PMA phorbol ester
  • MCV therapeutic vaccines Vac/G+I, Vac/R, Vac/C and Vac/M can induce antigen-specific cellular immune responses, especially antigens induced by Vac/R vaccine
  • the specific cellular immune response was the most significant, and the Vac/R vaccine had the most significant inhibitory effect on tumors.
  • MCV therapeutic vaccine prepared with GM-CSF+IFN- ⁇ , CpG1826, R848 or MPL as adjuvant can have certain anti-tumor effect.
  • the mechanism of action of MCV therapeutic vaccine was preliminarily discussed. Studies have found that MCV therapeutic vaccines can induce antigen-specific T cell immune responses. These results demonstrate the feasibility of the MCV therapeutic vaccine developed in this study, and also provide some data support for the clinical research of MCV therapeutic vaccine.
  • sVP1 soluble VP1
  • VP1 reassembled VP1
  • sVP1 and rVP1 were adsorbed on Al(OH) 3 adjuvant and named sVac and rVac, respectively.
  • BALB/c mice were intramuscularly injected on the 0th and 14th day, and the orbital blood was collected on the 28th day, and the serum was separated by centrifugation at 6000 rpm for 10 minutes, and the antigen-specific antibody titer was detected by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • sVP1 protein was diluted with antigen coating solution to 2 ⁇ g/mL, and 96-well ELISA plate was coated with diluted sVP1, 100 ⁇ L/well, and incubated at 4°C overnight.
  • Color development shake off the secondary antibody incubation solution. Washed 5 times with 1 ⁇ PBST. Add TMB substrate chromogenic solution, 100 ⁇ L/well, and incubate at 37°C for 5 minutes in the dark.
  • Stop add 2M sulfuric acid (H 2 SO 4 ) to stop the color reaction, 50 ⁇ L/well.
  • Antigen-specific antibody titers induced by vaccines sVP1-VAC or rVP1-VAC prepared with sVP1 or rVP1 antigens are shown in FIG. 19 .
  • the MCV therapeutic vaccine was applied to the CMS5-VP1 mouse tumor model and could achieve the effect of complete tumor regression.
  • BALB/c mice were inoculated with breast cancer cells 4T1 To establish a 4T1 mouse tumor model, and use the MCV therapeutic vaccine on this mouse model to test whether it has antigen specificity.
  • CpG1826 was synthesized by Shanghai Jierui Biotechnology; R848 was purchased from invivogen (tlrl-r848); aluminum hydroxide [Al(OH) 3 ] was purchased from Brenntag.
  • mice 6-8 week-old female BALB/c mice used in the experiment were purchased from Shanghai Sipple-Bike Laboratory Animal Co., Ltd., and were raised in Shanghai Sipple-Bike Laboratory Animal Co., Ltd. SPF clean mice All animal experiments were carried out under the guidance of animal protection guidelines.
  • BALB/c mice were injected subcutaneously with 1 ⁇ 10 6 /100 ⁇ L CMS5-VP1 on the left side and 1 ⁇ 10 6 /100 ⁇ L breast cancer cells 4T1 or fibrosarcoma cells CMS5 on the right side, respectively.
  • the tumor-bearing mice were randomly divided into five groups, and the MCV therapeutic vaccine (VP1/CpG/R848/Al(OH) 3 ) or VP1-free vaccine (CpG/R848/Al(OH) 3 ) or PBS.
  • MCV therapeutic vaccine VP1/CpG/R848/Al(OH) 3
  • VP1-free vaccine CpG/R848/Al(OH) 3
  • the tumor growth curve of CMS5-VP1 in tumor-bearing mice is shown in FIG. 20 .
  • the 4T1 tumor growth curve of tumor-bearing mice is shown in FIG. 21 .
  • Figure 22 shows the anti-tumor effects of MCV therapeutic vaccine on three tumor models of CMS5-VP1, CMS5 and 4T1.
  • CpG1826 was synthesized by Shanghai Jierui Biotechnology; R848 was purchased from invivogen (tlrl-r848); aluminum hydroxide [Al(OH) 3 ] was purchased from Brenntag.
  • mice 6-8 week-old female BALB/c mice used in the experiment were purchased from Shanghai Sipple-Bike Laboratory Animal Co., Ltd. All animal experiments were carried out under the guidance of animal protection guidelines.
  • mice On the fifth day after tumor inoculation, tumor-bearing mice were randomly divided into groups of 8 mice, and the vaccine was intramuscularly injected on the fifth and 12th day after tumor inoculation, respectively. Mice were divided into 5 groups, including:
  • VP1/CpG/Al(OH) 3 rVP1 10 ⁇ g; CpG 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • VP1/R848/Al(OH) 3 rVP1 10 ⁇ g; R848 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • VP1/CpG/R848/Al(OH) 3 rVP1 10 ⁇ g; CpG 10 ⁇ g; R848 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • VP1/Al(OH) 3 rVP1 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • mice On the fifth day after tumor inoculation, tumor-bearing mice were randomly divided into groups of 8, and the vaccine was intramuscularly injected on the 5th, 12th, and 19th day after tumor inoculation, respectively. Mice were divided into 4 groups including:
  • CpG/R848/Al(OH) 3 CpG 10 ⁇ g; R848 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • VP1/CpG/R848/Al(OH) 3 rVP1 10 ⁇ g; CpG 10 ⁇ g; R848 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • VP1/Al(OH) 3 rVP1 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • CM5-VP1 tumor cell rechallenge 30 days after the last immunization (2 injections: day 42; 3 injections: day 49), mice in the MCV vaccine-immunized group were subcutaneously injected with 1 ⁇ 10 6 /100 ⁇ L CMS5- VP1 cells.
  • CMS5-VP1 tumor rechallenge experiment tumor growth curve after the last immunization two needles on the left and three needles on the right.
  • MCV therapeutic vaccines can induce antigen-specific cellular immune responses in the body to achieve anti-tumor effects. Tumor growth curve of mice after vaccine immunization.
  • CpG1826 was synthesized by Shanghai Jierui Biotechnology; R848 was purchased from Invivogen (tlrl-r848); Aluminum hydroxide [Al(OH) 3 ] was purchased from Brenntag.
  • Mouse antibodies anti-CD3, anti-CD4, and anti-CD8 were purchased from BioxCell.
  • mice 6-8 week-old female BALB/c mice used in the experiment were purchased from Shanghai Sipple-Bike Laboratory Animal Co., Ltd. All animal experiments were carried out under the guidance of animal protection guidelines.
  • mice immunization On the 4th day after tumor inoculation, the tumor-bearing mice were randomly divided into 5 groups, with 5 mice in each group. Three groups of tumor-bearing mice were injected intraperitoneally on the 4th, 11th, and 18th days after tumor inoculation, respectively. 200 ⁇ g anti-CD3, anti-CD4, anti-CD8 monoclonal antibody to block CD3, CD4, CD8 T cells, 24 hours after antibody injection (5, 12, 19 days after tumor inoculation), intramuscular injection of MCV therapeutic vaccine (VP1/ CpG/R848/Al(OH) 3 ) or PBS.
  • MCV therapeutic vaccine VP1/ CpG/R848/Al(OH) 3
  • CpG1826 was synthesized by Shanghai Jierui Biotechnology; R848 was purchased from invivogen (tlrl-r848); aluminum hydroxide [Al(OH) 3 ] was purchased from Brenntag.
  • mice 6-8 week-old female BALB/c mice used in the experiment were purchased from Shanghai Sipple-Bike Laboratory Animal Co., Ltd. All animal experiments were carried out under the guidance of animal protection guidelines.
  • mice immunization On the fifth day after tumor inoculation, the mice were randomly divided into three groups, with five mice in each group, and the mice were immunized intramuscularly on the 5th, 12th, and 19th days respectively. Each group was based on the following components:
  • VP1/CpG/R848/Al(OH) 3 rVP1 10 ⁇ g; CpG 10 ⁇ g; R848 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • VP1/Al(OH) 3 rVP1 10 ⁇ g; Al(OH) 3 500 ⁇ g; 0.1 mL;
  • Figure 24 shows the expression levels of Treg in the lymph nodes of tumor-bearing mice in each vaccine group.

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Abstract

一种预防和治疗默克尔细胞癌的新型疫苗。通过将默克尔细胞癌多瘤病毒衣壳蛋白VP1作为抗原，结合TLR激动剂，构建了基于默克尔细胞癌多瘤病毒衣壳蛋白VP1的治疗性疫苗。

Description

一种预防和治疗默克尔细胞癌的新型疫苗 技术领域
本发明属于免疫学和病毒学领域，具体涉及一种预防和治疗默克尔细胞癌的新型疫苗。
背景技术
默克尔细胞癌多瘤病毒(MCV)是目前唯一的一种明确已知与人类癌症直接相关的多瘤病毒。默克尔细胞癌多瘤病毒是一种双链无包膜的DNA病毒，具有二十面体的约45nm的球状结构，基因组大小为5386bp，含有早期编码区、晚期编码区以及非编码调节区(NCRR)。默克尔细胞癌多瘤病毒的早期基因主要编码LT、ST、57kT以及ALTO(大T开放阅读框的交替框架)，而晚期基因主要编码衣壳蛋白VP1以及VP2。VP1具有启动病毒感染的作用，常以五聚体的形式存在，72个五聚体可以在真核***内自组装成病毒样颗粒。
默克尔细胞癌是一种罕见的侵袭性皮肤癌，好发于暴露在阳光的头颈等部位，表现为快速生长的***结节。大约80％的默克尔细胞癌(MCC)病人肿瘤内能检测到整合至肿瘤细胞内的默克尔细胞癌多瘤病毒。同时，约60％-80％健康人群中能检测到默克尔细胞癌多瘤病毒衣壳蛋白VP1的特异性抗体。默克尔细胞癌多瘤病毒主要以无症状的方式感染人类幼年期，紫外照射，疾病或年龄增长导致的免疫力降低，均是默克尔细胞癌发病的诱因。默克尔细胞癌发病率不高，但是其临床预后极差，是致死率最高的皮肤源性肿瘤之一。最常用的治疗手段是通过化疗或手术切除，然而其复发率高达40％。近年来，随着免疫检查点抑制剂(PD-L/PD-L1抑制剂)的兴起和广泛应用，部分单抗已可应用于晚期转移性默克尔细胞癌的治疗中，但该治疗方法极大依赖病人自身免疫力，对于不同个体来说反应率相差较大。疫苗作为医学史上最成功的疾病干预措施，其重要性不言而喻，而重组病毒样颗粒(VLP)疫苗，由于其不含有病毒遗传物质，但具有类似于天然病毒的形态结构，因此兼具了安全性以及良好的免疫原性。从而作为预防性疫苗被应用到了各种领域，例如人类***状瘤病毒(HPV)，甲型/乙型/戊型肝炎病毒等。与此同时，也有动物实验表明，病毒样颗粒疫苗能够在不施用任何佐剂的情况下对已建立的***状瘤起到治疗效果。基于此，对于能够自组装成病毒样颗粒的默克尔细胞癌多瘤病毒VP1来说，基于该蛋白的相关疫苗的研发，在默克尔细胞癌的预防或治疗方面显示出了巨大的潜力。
现有技术中，对于不同病毒的VP1已经进行了相关疫苗的研发。
例如：中国专利200810232479.6公开了一种B19病毒VP1独特区基因，并构建了B19原核表达pQE30-VP1独特区克隆质粒、表达并纯化出重组蛋白，该重组蛋白体外能够引起免疫细胞应答，产生高效价的抗体。
例如：中国专利201010126145.8公开了一种密码子优化的EV71VP1基因及其核酸疫苗，该疫苗能够在真核细胞293T细胞中有效表达，免疫动物能刺激产生特异性VP1抗体。但是该疫苗仅仅采用了核酸作为疫苗的有效成分，免疫效果较差。
对于默克尔细胞癌多瘤病毒感染细胞或默克尔细胞癌癌细胞的清除来说，中和抗体与体内CD4 +/CD8 +的T细胞的协同作用是至关重要的。虽然默克尔细胞癌患者中观察到的默克尔细胞癌多瘤病毒VP1特异性抗体滴度高于健康人，但相关临床数据表明，高水平的抗VP1抗体预示着更好的临床预后。
基于默克尔细胞癌多瘤病毒的广泛感染率、默克尔细胞癌的高致死率，对其进行进一步深入研究是极为重要且具有现实意义的。
发明内容
术语
除非另外定义，否则本文使用的所有技术和科学术语的含义与本发明所属领域的普通技术人员通常理解的含义相同。
本文使用的以下术语则具有如下含义。
在本文和所附权利要求中所使用的单数形式“一”，“一个”和“该”，除非上下文另有明确说明，均包括复数指代。同样，术语“一”(或“一个”)，“一个或多个”和“至少一个”在本文中可互换使用。术语“包含”，“包括”和“具有”可以互换使用。
本文所使用的术语“疫苗”是指可以施用于人或动物以诱导免疫***反应的组合物；这种免疫***反应可以导致抗体的产生或仅激活某些细胞，特别是抗原呈递细胞，T淋巴细胞和B淋巴细胞。疫苗组合物可以是用于预防目的或用于治疗目的的组合物，或同时用于预防目的及治疗目的的组合物。
本文所用的术语“施用”，是指在用疫苗或某种组合物治疗患者的领域中它们的通常和普遍的含义。本文所用术语“共同施用”和“伴随施用”是同义词，并且是指以这样的方式给患者施用两种物质或两种组合物，使得两种物质或两种组合物都存在于患者体内。同时，共同施用可以是同时的或有序的，并且共同施用的物质或组合物可以同时施用于患者，或单独但时间接近，或在同一天施用，或以其他能使体内各物质或组合物的停留时间显着重叠的方式施用于患者。给药，非肠胃注射给药，可包括皮下给药，肌肉给药，皮内给药，腹膜内给药，眼内给药，鼻内给药和静脉内给药。
本发明涉及到的疫苗或组合物可以根据本领域已知的方法施用于个体。这些方法包括非肠胃给药，例如通过皮肤注射或注射到皮肤的所有注射途径：比如，肌肉内，静脉内，腹膜内，皮内，粘膜，粘膜下或皮下等。此外，疫苗可作为滴剂，喷雾剂，凝胶剂或软膏剂局部施用于眼，鼻，口，***或***的粘膜上皮，或施用于身体任何部位的外表皮上。其他可能的施用途径是通过呼吸道吸入喷雾，气溶胶或粉末。在最后一种情况下，使用的颗粒大小将决定颗粒进入呼吸道的深度。或者，可以通过消化途径，以粉末、液体或片剂的形式与食物，饲料或饮用水结合施用，或通过液体，凝胶，片剂或胶囊的形式直接施用于口腔，或通过栓剂的形式施用于***。在一个实施例中，可以通过使用电脉冲仪或任何电穿孔装置递送DNA疫苗，以促进DNA疫苗渗入宿主细胞并有效表达。
当在数字表达前使用术语“约”时(比如温度、时间、量和浓度，包括范围)，表示可以是(+)或(-)10％，5％或1％的近似值。
本文所用的术语“针对”，在疫苗领域指“治疗”、“预防”、“辅助治疗”或同等效能的词语及其组合。
本文所用术语“针对默克尔细胞癌”应当被不限制性地理解为针对默克尔细胞癌多瘤病毒。
本文所用术语“治疗”和“预防”以及由此产生的词汇不一定意味着100％或完全治疗或预防。相反，本领域某些技术手段不同程度的治疗或预防被认为具有潜在的益处或治疗效果。在一个实施例中，本发明的组合物或方法可以在哺乳动物中提供任何剂量、任何水平的疾病治疗或预防。此外，本发明方法提供的治疗或预防可包括治疗或预防疾病、癌症的一种或多种病症或症状。而且，出于本文的目的，“预防”可以包括延迟疾病的发作或其症状或病症。关于本发明的方法，癌症可以是任何癌症，包括与本文所述的任何肿瘤抗原相关的任何癌症。
本文所用术语“佐剂”是指当与制剂中的特定免疫原(例如基于VLP的疫苗)组合使用时，将增强或减少性地改变或修饰免疫应答。免疫应答的修饰包括增强或扩大特异性抗体或细胞免疫应答，一种或二者共同均包含在范围之内。免疫应答的修饰还可以指减少或抑制某些抗原特异性免疫应答。
本文所使用的术语“病毒样颗粒”或“VLP”是指非复制型的病毒壳颗粒。病毒结构蛋白(比如包膜或衣壳蛋白)在特定条件下的表达可导致VLPs的自组装。VLPs通常由一种或多种病毒蛋白组成，例如包括但不限于被称为衣壳、外壳、壳、表面和/或包膜蛋白的那些蛋白，或衍生自这些蛋白的颗粒形成的多肽。在适当的表达***中重组表达蛋白后，VLPs可以自发形成。产生特定VLPs的方法在本领域已知的，下面将更全面地讨论。病毒蛋白重 组表达后VLPs的存在可以使用本领域已知的常规技术进行检测，例如通过电子显微镜，生物物理表征等。参见，Baker et al.,Biophys.J.(1991)60:1445-1456；Hagensee et al.,J.Virol.(1994)68:4503-4505。例如，VLPs可以通过密度梯度离心分离和/或通过特征密度条带鉴别。或者，可以使用低温电子显微镜对所讨论的VLP制剂的玻璃化水性样品进行检查，并在适当的曝光条件下记录图像。VLP纯化的其他方法包括但不限于色谱技术，例如亲和，离子交换，尺寸排阻和反相过程。此外，任何纳米测量装置均可用于测量纳米颗粒的尺寸。
本发明还涉及到多核苷酸和多肽的变异型。本文所用术语“变异型”是指不同于本发明中的多核苷酸或多肽但保留其基本特性的多核苷酸或多肽。通常，变异型总体上非常相似，并且在许多区域中，与本发明的多核苷酸或多肽相同。
变异型可以包含编码区，非编码区任其一或两者共同的改变。大多倾向于产生沉默取代，添加或缺失，但不改变编码多肽的性质或活性的多核苷酸变异型。其中较倾向于由于遗传密码的简并性而由沉默取代产生的核苷酸变异型。此外，5-10，1-5或1-2个氨基酸以任何组合被取代，缺失或添加的变异型也常出现。
一个变异型的实例为截短，一个截短的实例为MCV-VP1截短可包括C-末端55aa(369-423)缺失。来自野生型MCV-VP1的C'-缺失可以产生具有aa1-369片段的MCV-VP1截短，其可以与其他抗原一起用作于嵌合VLP的融合蛋白。
一个变异型的实例为缺失，一个缺失的实例为MCV-VP1的C末端17aa(352-368)缺失。
一个变异型的实例为突变，一个突变的实例为MCV-VP1的E353突变，由L或F替换。
本发明还包括所述多核苷酸的等位基因变异型。等位基因变异型表示占据相同染色体基因座的基因的两种或更多种替代形式中的任何一种。等位基因变异型通过突变自然产生，并可能导致群体内的多态性。基因突变可以是沉默的(编码的多肽中没有变化)或可以编码具有改变的氨基酸序列的多肽。多肽的等位基因变异型是由等位基因变异型编码的多肽。
本发明还涉及含有MCV VP1的非编码区或与其杂交的多核苷酸片段。多核苷酸片段可以是与天然存在的核苷酸长度相等或更短的多核苷酸片段。此类片段可用于诊断。常出现含有剪切点或与含有剪切点的多核苷酸杂交的多核苷酸片段。可以使用任何长度如50，150，500，600，1000，1100或约1260个核苷酸的多核苷酸片段。
本发明通过将优化后的默克尔细胞癌多瘤病毒(MCV)结构蛋白VP1的cDNA序列在真核***和原核***中克隆，在原核***中表达VP1蛋白，构建基于VP1的，针对默克尔细胞癌多瘤病毒的DNA和/或蛋白疫苗。本发明提供的单独免疫针对默克尔细胞癌的DNA疫苗或是针对默克尔细胞癌的蛋白疫苗可以诱导细胞免疫反应或高水平的抗VP1抗体水平。本研究是第一项使用VP1作为针对默克尔细胞癌多瘤病毒的疫苗抗原的研究，为针对默克尔细胞癌的疫苗研究奠定了基础。
除非特别指明，本文中的“Vehicle”均指“PBS”,“OVA”均指“卵清白蛋白”。
除非特别指明，本文中“VP1”或“重组蛋白VP1”是指“重组默克尔细胞癌多瘤病毒衣壳蛋白”，“rVP1”或“重组装或自组装VP1”是指“重组装或自组装默克尔细胞癌多瘤病毒衣壳蛋白”。
一方面，本发明提供了一种针对默克尔细胞癌的疫苗组合物，其特征在于，所述疫苗组合物包含默克尔细胞癌多瘤病毒衣壳蛋白VP1或者包含编码默克尔细胞癌多瘤病毒衣壳蛋白VP1的基因的质粒，以及TLR激动剂，所述TLR激动剂选自CpG、R848和MPL中的一种或多种。其中，包含默克尔细胞癌多瘤病毒衣壳蛋白VP1的为蛋白疫苗组合物。其中，包含编码默克尔细胞癌多瘤病毒衣壳蛋白VP1的基因的质粒的为DNA疫苗组合物。
在本发明的一些优选方案中，所述TLR激动剂为CpG和R848的组合。
在本发明的一些优选方案中，所述组合物进一步包含铝佐剂。在本发明的一些优选方案 中，所述疫苗包括5-500重量份的默克尔细胞癌多瘤病毒衣壳蛋白VP1蛋白或包含编码默克尔细胞癌多瘤病毒衣壳蛋白VP1的基因的质粒、2.5-250重量份的TLR激动剂和10-1000重量份的铝佐剂。
在本发明的一些优选方案中，所述疫苗包括10-100重量份的默克尔细胞癌多瘤病毒衣壳蛋白VP1蛋白或包含编码默克尔细胞癌多瘤病毒衣壳蛋白VP1的基因的质粒、5-100重量份的TLR激动剂和50-500重量份的铝佐剂。
在本发明的一些优选方案中，所述的VP1的核苷酸序列与SEQ ID No.6具有71.9-74.7％同源性。
在本发明的一些优选方案中，所述的VP1的核苷酸序列为SEQ ID No.1、SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示。
另一方面，本发明提供了一种默克尔细胞癌多瘤病毒衣壳蛋白VP1的编码基因。
具体地，所述的编码基因与SEQ ID No.6具有71.9-74.7％同源性的核苷酸序列。
具体地，所述的编码基因为SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示的核苷酸序列。
另一方面，本发明还提供了上述默克尔细胞癌多瘤病毒衣壳蛋白VP1的编码基因在制备针对默克尔细胞癌的疫苗中的应用。
具体地，所述的应用为利用与SEQ ID No.6具有71.9-74.7％同源性的核苷酸序列或其变异型中的一种或多种制备针对默克尔细胞癌的DNA疫苗。
具体地，所述的应用为利用SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示的核苷酸序列或其变异型中的一种或多种制备针对默克尔细胞癌的DNA疫苗。
具体地，所述的应用为利用与SEQ ID No.6具有71.9-74.7％同源性的核苷酸序列和/或其变异型编码的蛋白和/或其突变体中的一种或多种制备针对默克尔细胞癌的蛋白疫苗。
具体地，所述的应用为利用SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示的核苷酸序列和/或其变异型编码的蛋白和/或其突变体中的一种或多种制备针对默克尔细胞癌的蛋白疫苗；进一步具体地，所述的蛋白包括但不限于SEQ ID No.5的氨基酸序列。
具体地，所述的应用为同时利用与SEQ ID No.6具有71.9-74.7％同源性的核苷酸序列和/或其变异性及其编码的蛋白和/或其突变体中的一种或多种制备针对默克尔细胞癌的疫苗。
具体地，所述的应用为同时利用SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示的核苷酸序列和/或其变异型及以上核苷酸序列编码的蛋白和/或其突变体中的一种或多种制备针对默克尔细胞癌的疫苗。
进一步具体地，所述的应用括但不限于利用SEQ ID No.5的氨基酸序列制备针对默克尔细胞癌的疫苗。
再一方面，本发明提供了一种针对默克尔细胞癌的疫苗。
具体地，所述的疫苗为DNA疫苗。
具体地，所述的DNA疫苗包含与SEQ ID No.6具有71.9-74.7％同源性的核苷酸序列和/或其变异型中的一种或多种。
具体地，所述的DNA疫苗包含SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4的核苷酸序列和/或其变异型中的一种或多种。
具体地，所述DNA疫苗构建到了载体上，所述的载体包含但不限于pVAX1载体，pcDNA3.1，NTC8385，或者病毒载体，如腺病毒Ad载体，腺相关病毒AAV载体等。
具体地，所述的蛋白疫苗包含与SEQ ID No.6具有71.9-74.7％同源性的核苷酸序列和/或其变异型所编码的蛋白和/或其突变体中的一种或多种。
具体地，所述的蛋白疫苗包含SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4和/或其变异型核苷酸序列编码的蛋白和/或其突变体中的一种或多种。
进一步具体地，所述的SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4编码的蛋白质包含但不限于SEQ ID No.5的氨基酸序列。
作为一种推荐的且非限制性的施用方法，所述的DNA疫苗的注射量为1-200μg/只小鼠。
作为一种推荐的且非限制性的制备方法，所述蛋白疫苗可以利用大肠杆菌***进行制备。产生VP1蛋白，形成不同大小的不含DNA的病毒样颗粒(VLPs)，例如，通过自我装配。
作为一种推荐的且非限制性的制备方法，所述蛋白疫苗可以利用其他生物类的蛋白表达***进行制备。包括但不限于酵母表达***，杆状病毒表达***，中国仓鼠卵巢细胞(CHO)表达***等。
作为一种推荐的且非限制性的制备方法，所述的蛋白疫苗中的病毒样颗粒在形成蛋白疫苗前进行了纯化。
作为一种推荐的且非限制性的制备方法，VP1蛋白病毒样颗粒的大小在约1nm-约1μm，约5nm–约800nm，约8nm–约700nm，约10nm–约400nm，约20nm–约200nm，约25nm–约100nm，或者约30nm–约50nm之间；在一个实施例中，VP1蛋白的病毒样颗粒大小更加集中在约20nm–约200nm，约25nm–约100nm，或者约30nm–约50nm之间。
所述的疫苗还包含佐剂，所述佐剂包含TLR激动剂和铝佐剂，所述TLR激动剂选自CpG、R848和MPL中的一种或多种。
优选地，所述疫苗包括5-500重量份的DNA疫苗或蛋白疫苗、2.5-250重量份的TLR激动剂和10-1000重量份的铝佐剂。
进一步优选地，所述疫苗包括10-100重量份的DNA疫苗或蛋白疫苗、5-100重量份的TLR激动剂和50-500重量份的铝佐剂。
作为一种推荐的且非限制性的施用方法，所述蛋白疫苗的注射量为1.1-20μg/只小鼠。
具体地，所述的疫苗为预防性和/或治疗性疫苗。
具体地，本发明提供的疫苗适用于任何哺乳动物。所述的哺乳动物是指温血脊椎动物，包括但不限于人、兔、猫、犬、猪、牛和狗。
具体地，本发明涉及到的疫苗或包含其的产品的剂型包括但不限于滴剂、喷雾剂、凝胶剂、粉剂、软膏剂、片剂、颗粒剂。
附图说明
图1为基础实验例1中的CMS5-VP1细胞系肿瘤生长曲线图。
图2为实施例3制备的VP1蛋白疫苗检测结果。
图3为实施例6中的纯化后的VP1蛋白SDS-PAGE检测结果。
图4为实施例7中的Nu-PAGE检测不同组装工艺下形成的VP1结构蛋白结果。
图5为实施例8中的蛋白疫苗VP1免疫原性验证中小鼠特异性MCV-VP1抗体滴度以及DTH(迟发型超敏反应)、细胞免疫结果。
图6为实施例9中的DNA疫苗的免疫反应检测结果。
图7为实施例10中的联合免疫终免后7天的DTH及特异性抗体滴度结果。
图8为实施例11肿瘤接种后肿瘤小鼠肿瘤生长曲线，其中箭头位置为疫苗免疫时间点。
图9为实施例12肿瘤接种后第33天各组小鼠荷瘤图片。
图10为实施例12肿瘤接种后第33天各组荷瘤小鼠肿瘤组织。
图11为实施例12肿瘤接种后各组荷瘤小鼠肿瘤生长曲线。
图12为实施例13中的注射rVP1或PBS后24小时的脚掌肿胀程度。
图13为实施例13中的含有佐剂的MCV-VP1治疗性肿瘤疫苗后对小鼠DTH的实验结果。
图14为实施例13中的含有佐剂的治疗性疫苗免疫后的小鼠VP1抗体滴度结果。
图15为实施例13中的含有佐剂的治疗性疫苗免疫后的小鼠CD4 +、CD8 +T细胞的TNF-α、IFN-γ及IL-2表达水平的流式检测结果。
图16为实施例13中肿瘤接种19、34天后各组小鼠体内肿瘤体积。
图17为实施例13中肿瘤接种19天后小鼠脾细胞经VP1刺激后，通过流式细胞术检测CD4 +、CD8 +T细胞的TNF-α、IFN-γ及IL-2分泌水平结果。
图18为实施例13中肿瘤接种34天后小鼠脾细胞经VP1刺激后，通过流式细胞术检测CD4 +、CD8 +T细胞的TNF-α、IFN-γ及IL-2分泌水平结果。
图19为实施例14中以sVP1或rVP1位抗原制备的疫苗sVP1-VAC或rVP1-VAC诱导的抗原特异性抗体滴度。
图20为实施例15中荷瘤小鼠CMS5-VP1肿瘤生长曲线。
图21为实施例15中荷瘤小鼠4T1肿瘤生长曲线。
图22为实施例15中MCV治疗性疫苗对CMS5-VP1、CMS5、4T1三种肿瘤模型的抗肿瘤效果。
图23为实验例17中MCV治疗性疫苗免疫后小鼠肿瘤生长曲线
图24为实施例18中各疫苗组荷瘤小鼠***中Treg的TGF-β表达水平。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
基础实验例1CMS5-VP1肿瘤细胞株构建、筛选、验证
CMS5-VP1肿瘤细胞株将主要应用于疫苗效果的验证。
1、pcDH-VP1质粒构建
(1)首先根据pcDH-GFP质粒设计引物：
MCV-F(5’-CTAGAGCTAGCGTTTAAACTTAAGC-3’)；
MCV-R(5’-GCGGCCGCTCTAGACTCGAGTCACAGCTCCTG-3’)。
其中MCV-F的5’端含有一个Nhe I酶切位点，MCV-R的3’端含有一个Not I酶切位点。
(2)PCR扩增
以质粒pVAX1-VP1为模板，扩增得到在N端、C端分别含有内切酶位点的VP1片段。PCR反应体系见下表1。
表1 PCR反应试剂用量表
Figure PCTCN2021127386-appb-000001
PCR反应程序如下表2。
表2 PCR反应程序
Figure PCTCN2021127386-appb-000002
(3)酶切反应
PCR扩增的VP1片段及载体pCDH以内切酶NheI/Not I于37℃水浴锅中酶切4h后回收VP1片段及pcDH载体。酶切体系见下表3。
表3 双酶切反应体系表
Figure PCTCN2021127386-appb-000003
(4)酶连、转化反应
酶切后回收的VP1及pcDH片段产物根据摩尔质量比1:3的比例以T4连接酶室温连接反应40分钟。
连接产物通过化学转化法转化至大肠杆菌克隆宿主菌DH5α感受态细胞中。具体方法为：
1)连接产物与50μL DH5α感受态细胞混匀，冰浴30分钟。
2)42℃热击90秒。
3)冰上放置3分钟。
4)加入500μL无抗生素的LB液体培养基。
5)37℃摇床以150rpm振荡培养45分钟。
6)13000rpm离心，弃上清。
7)以100μL无抗生素的LB液体培养基重悬菌体，涂布于含氨苄青霉素(50μg/mL Amp)的LB平板上，37℃培养箱倒置培养过夜，标记为pcDH-VP1/DH5α。
(5)菌落PCR鉴定及测序
从过夜培养的平板随机挑选6个pcDH-VP1/DH5α单克隆菌落以2×PCR Mix进行PCR鉴定。具体方法如下：
1)分别取10μL 2×PCR Mix于无菌的PCR管中，分别加入0.2μL的引物MCV-F、MCV-R。
2)以10μL的Tip从平板上挑取单克隆菌落，以移液枪吹打混匀于11μL无菌的去离子水中。
3)取一个新的LB(50μg/mL Amp)平板，在平板底部画好格子，标好克隆号，37℃培养箱倒置培养10小时。
4)将吹打均匀的pcDH-VP1/DH5α菌液分别点于LB平板对应的位置上。
5)将剩余的pcDH-VP1/DH5α菌液加入至含有引物的2×PCR Mix中，进行PCR反应，PCR反应条件如前述步骤1中(2)PCR扩增条件。
6)PCR结束后，1％琼脂糖凝胶电泳检测PCR产物。
(6)测序鉴定
根据PCR结果，挑取PCR阳性的克隆，于5mL LB液体培养基(50μg/mL Amp)中，37℃振荡培养过夜。
过夜培养的pcDH-VP1/DH5α单克隆菌液各取1mL，以通用引物CMV-F和EF1α-R测序。剩余的菌液与60％的无菌甘油以3:1的比例混匀，-20℃冻存，备用。
根据测序结果进行序列比对，确定序列正确的克隆号，将序列正确的甘油菌冻存于-80℃。
(7)pcDH-VP1质粒制备
取1支-80℃冻存的pcDH-VP1/DH5α1:100接种于200mL LB液体培养基(50μg/mL Amp)中，37℃摇床220rpm振荡培养过夜，以去内毒素质粒大抽试剂盒(QIAGEN,12362)抽提pcDH-VP1质粒。
实验结果：
以质粒pVAX1-VP1为模板PCR扩增得到的PCR产物取1μL，以1％琼脂糖凝胶电泳进行检测，PCR扩增得到的目的条带大小与理论大小一致。通过PCR产物回收试剂盒(QIAGEN公司，28104)回收VP1片段。
回收到的VP1片段及载体pcDH-GFP以Nhe I/Not I进行酶切反应，酶切产物以1％琼脂糖凝胶电泳。
紫外灯下将含VP1及pcDH的凝胶条带切下，以胶回收试剂盒(QIAGEN公司，28115)回收目的片段。回收得到的VP1及pcDH按下表4的反应体系进行酶连反应。
表4 酶连反应体系表
Figure PCTCN2021127386-appb-000004
酶连反应后转化至大肠杆菌DH5α感受态细胞中，标记为pcDH-VP1/DH5α。随后随机挑取10个单克隆菌落开展的菌落PCR。为了进一步确定构建的pcDH-VP1，将阳性克隆进行测序鉴定挑选序列正确克隆。
取pcDH-VP1/DH5α甘油菌1:100接种于200mL LB(50μg/mL)液体培养基中37℃振荡培养过夜，抽提的质粒用于构建CMS5-VP1细胞系用。
2、CMS5-VP1细胞系构建
CMS5细胞是药物诱导BALB/c小鼠产生的纤维肉瘤细胞系。CMS5细胞为贴壁细胞。CMS5细胞自身不耐嘌呤霉素(Puromycin)，载体pcDH携带的Puromycin抗性基因整合到细胞基因组而赋予了细胞的Puromycin耐药性。因此，通过脂质体转染法将pcDH-VP1转染至CMS5细胞中，在Puromycin药物压力下，未转染pcDH-VP1的CMS5细胞大量死亡，从而筛选得到表达VP1的细胞系CMS5-VP1。
(1)Puromycin药物浓度确定
为了确定Puromycin药物对CMS5的杀伤剂量，以含不同浓度Puromycin的RPMI1640(10％FBS，1％双抗)完全培养基中培养CMS5细胞。24小时后，以台盼蓝染色，在显微镜下观察细胞的死亡率来确定CMS5细胞100％致死率的药物浓度作为CMS5-VP1细胞系筛选的药物压力。具体方法如下：
1)冻存于液氮罐的CMS5细胞在37℃水浴锅中解冻后，加入10mL的RPMI1640无血清培养基，1000rpm离心5分钟。
2)去除上清液，以10mL RPMI1640完全培养基重悬细胞，于10cm细胞培养皿中5％CO 2、37℃培养箱培养。
3)待CMS5细胞生长至占培养皿80％时，去除培养基，加入2mL无菌1×PBS将培养皿浸没。
4)去除PBS，加入1mL胰酶，5％CO 2、37℃培养箱消化30秒。
5)加入5mL RPMI1640完全培养基终止胰酶的消化作用后，1000rpm离心5分钟。
6)去除上清液，以5mL RPMI1640完全培养基重悬细胞后，取2.5mL细胞悬液与7.5mL RPMI1640完全培养基混匀，于10cm细胞培养皿中，5％CO 2、37℃培养箱培养。此即为细胞复苏及1:2传代。
7)如此传代4次后，状态良好的CMS5细胞以胰酶消化后，以RPMI1640完全培养基制备成细胞悬液。
8)CMS5细胞悬液以1×10 6/孔铺于12孔细胞培养板中，5％CO 2的37℃培养箱中培养过夜。
9)细胞贴壁后，将培养基更换为含有0.25μg/mL、0.5μg/mL、1μg/mL、2μg/mL、5μg/mL、10μg/mL及20μg/mL Puromycin的RPMI1640完全培养基。培养24小时后，胰酶消化的CMS5细胞，以台盼蓝染色，显微镜下计算细胞的致死率以确定最终的Puromycin药物浓度。
(2)脂质体转染法转染pcDH-VP1
通过脂质体转染方法，将pcDH-VP1质粒转染至CMS5细胞中。转染48小时后，通过Puromycin药物压力来筛选带有目的质粒的细胞，通过长时间的药物筛选达到筛选稳定表达的细胞株。具体方法如下：
1)以RPMI1640完全培养基培养CMS5细胞于150mm培养皿中。
2)CMS5细胞生长至占培养皿80％时，胰酶消化细胞后，以无抗生素的RPMI1640完全培养基制备细胞悬液后，分别取1×10 7CMS5细胞铺于2个10cm细胞培养皿中，5％CO 2的37℃培养箱中培养过夜。
3)过夜培养的CMS5细胞大概占培养皿80％。
4)DNA(pcDH-VP1或pcDH-GFP质粒)与Lipofectamine2000以1:3的比例分别稀释于无血清OPTI-MEM中，室温静置20分钟。
5)将稀释的DNA(pcDH-VP1或pcDH-GFP)于稀释的Lipofectamine 2000以1:1的比例混匀，室温静置5分钟。
6)将DNA-Lipofectamine2000混合物加至过夜培养的CMS5培养皿中。
7)转染48小时后，将培养皿中培养基移除，加入含有Puromycin的RPMI1640完全培养基，5％CO 2、37℃培养箱中培养。转染前期，细胞大量死亡，每3天进行一次半换液。待细胞缓慢增殖后，每3天进行一次全换液。
8)待细胞第一次长满培养皿后，开始传代换液。
(3)CMS5-VP1细胞基因鉴定
细胞第一次长满传代后，取部分CMS5-VP1或CMS5-GFP细胞以试剂盒抽提RNA，通过RT-PCR扩增得到cDNA。
以扩增得到的cDNA为模板、以MCV-F1及MCV-R1为引物进行PCR扩增。
通过1％琼脂糖凝胶电泳检测PCR扩增结果。
(4)CMS5-VP1细胞株筛选
CMS5-VP1细胞经过6轮药物压力筛选后，开展单克隆细胞筛选。具体方法如下：
1)将CMS5-VP1细胞以胰酶消化后，台盼蓝染色，显微镜下以血球计数板计数。
2)根据细胞计数结果，以含Puromycin的RPMI1640完全培养基将CMS5-VP1细胞稀释至密度为5个/mL。
3)96孔细胞板加入CMS5-VP1细胞稀释液，100μL/孔以保证0.5个细胞/孔。5％CO 2、37℃培养箱中培养。
4)96孔培养板每三天换一次RPMI1640完全培养基(Puromycin)。
5)当细胞长成一个圆斑形态时，以胰酶消化细胞，再次稀释至密度5个/mL后于96孔细胞培养板中进行第二轮单克隆细胞筛选，每孔100μL(0.5个/孔)。
6)当细胞长成一个圆斑形态时，胰酶消化后，转移至24孔细胞培养板培养。
7)三天换一次培养基，第一次为半换液，直至长满后，以胰酶消化，转移至12孔细胞培养板中扩大培养。
8)三天换一次培养基，第一次为半换液，待12孔细胞培养板长满后转移至6孔板中培养。
9)待6孔细胞培养板长满后，取一半细胞以细胞冻存液(90％FBS、10％DMSO)冻存于-80℃。另一半细胞转移至10cm细胞培养皿中培养(三天换一次培养基)。细胞基本在第六天长满。
10)10cm细胞培养皿长满后，胰酶消化，取5×10 6细胞以细胞冻存液(90％FBS、10％DMSO)冻存于-80℃。
(5)CMS5-VP1细胞鉴定
经过两轮筛选得到的CMS5-VP1细胞株，分别取5×10 6细胞通过1000rpm离心5分钟收集细胞，通过免疫印迹检测VP1的表达。具体方法如下：
1)离心收集到的5×10 6细胞以1×PBS洗涤两次以去除残余培养基，1000rpm离心5分钟收集细胞。
2)以50μL的1×细胞裂解液重悬细胞，4℃静置2小时。
3)1000rpm离心5分钟，取上清。
4)加入10μL的6×loading buffer，煮沸10分钟。
5)12％的SDS-PAGE凝胶电泳(80V 30分钟,120V 60分钟)。
6)以PVDF膜进行转膜。
7)将转移了蛋白的PVDF膜放入5％脱脂奶粉的1×TBST封闭液中。室温下，水平摇床上低速封闭1h。
8)1×TBST洗涤3次，每次5分钟。
9)一抗孵育：抗VP1兔多抗1:5000稀释于5％脱脂奶粉的1×TBST中，PVDF膜置于一抗稀释液中，室温孵育1h。
10)1×TBST洗涤3次，每次5分钟。
11)二抗孵育：HRP标记的羊抗兔IgG 1:10000稀释于5％脱脂奶粉的1×TBST中，PVDF膜置于二抗稀释液中，室温孵育1h。
12)1×TBST洗涤5次，每次5分钟。
13)ECL显色(天能公司，180-501)：底物反应液1、2以1:1比例混匀，将PVDF膜倒扣于ECL反应液中，化学发光检测。
实验结果：
1、Puromycin药物浓度确定
CMS5细胞自身不耐Puromycin，而转入pcDH-VP1的CMS5-VP1由于载体pcDH携带一个Puromycin抗性的基因而具有Puromycin耐药性。为了确定CMS5细胞对Puromycin的耐药浓度，分别以含0.25μg/mL、0.5μg/mL、1μg/mL、2μg/mL、5μg/mL、10μg/mL及20μg/mL Puromycin的RMPI1640完全培养基培养CMS5细胞。培养24小时后，显微镜下观察细胞的状态。当Puromycin浓度达到5μg/mL时，细胞大量死亡。
为了进一步确定Puromycin对CMS5细胞的杀伤作用，以台盼蓝染色计算CMS5细胞的死细胞及活细胞数，当Puromycin达到5μg/mL时，CMS5细胞100％死亡。因此确定5μg/mL的Puromycin作为CMS5-VP1细胞筛选的药物浓度。
2、CMS5-VP1细胞筛选及鉴定
根据Lipofectamine2000操作手册，取质粒pcDH-VP1或pcDH-GFP 10μg，及30μL Lipofectamine2000分别稀释于500μL无血清OPTI-MEM中，室温静置5分钟后将稀释后的pcDH-VP1与Lipofectamine 2000混匀，室温静置20分钟。
将pcDH-VP1或pcDH-GFP与Lipofectamine 2000形成的DNA-脂质体复合物加至过夜培养的CMS5细胞中，混匀后，5％CO 2培养箱37℃培养
转染48小时后将培养基更换为5μg/mL Puromycin的RMPI1640完全培养基,在药物压力作用下，CMS5细胞大量死亡，CMS5-VP1细胞缓慢增殖。
为了确定CMS5转染是否成功，在构建CMS5-VP1细胞系的同时，以携带报道基因GFP的pcDH-VP1转染CMS5作为阳性对照。转染后第21天，以荧光显微镜观察CMS5-VP1或CMS5-GFP细胞增殖情况，分别在白光或GFP荧光下观察细胞。
在5μg/mL Puromycin的药物压力下，在荧光显微镜下观察到CMS5-GFP细胞表达的GFP蛋白。在荧光显微镜下未观察到CMS5-VP1细胞发出绿色荧光。这说明该方法筛选稳定表达目的基因的细胞株是可行的。
同时取1×10 7的CMS5-VP1或CMS5-GFP细胞抽提RNA并通过RT-PCR获得cDNA。为了确定VP1基因是否整合至CMS5细胞中，设计引物MCV-F1(5’-GTGGAGGTGCTGTCCGTGGTG-3’)及MCV-R1(5’-CAGGAAGCCCACGATATCGGC-3’)以扩增VP1的部分片段，大小为760bp。
以CMS5-VP1或CMS5-GFP cDNA为模板、MCV-F1及MCV-R1为引物进行PCR扩增，其中以质粒pcDH-VP1为阳性对照。
扩增得到的PCR产物以1％琼脂糖凝胶电泳检测目的基因VP1，CMS5-VP1 cDNA为模板扩增得到的条带大小与阳性对照大小一致。同时4号条带为CMS5 cDNA为模板的阴性对照，而5号条带为CMS5-GFP的非特异性扩增结果。
六轮的药物筛选后，将消化下来的CMS5-VP1细胞稀释后进行两轮的细胞筛选，免疫印迹鉴定得到5株表达VP1的CMS5-VP1细胞株。免疫印迹结果表明，CMS5-VP1/E2表达量最高，其次为E4E5。为了开展CMS5-VP1成瘤实验，选择了表达量低、中、高的细胞株CMS5-VP1/B5G2、F5及E2开展下一步的成瘤实验。
3、CMS5-VP1细胞成瘤实验
通过免疫印迹确定CMS5-VP1细胞株的VP1表达量，选择其中3株细胞开展成瘤实验。具体方法如下：
1)RPMI1640完全培养基(Puromycin)培养的CMS5-VP1细胞及RPMI1640完全培养基培养的CMS5细胞分别以胰酶消化，制备成细胞悬液。
2)台盼蓝染色后，显微镜下血球计数板计数。
3)以1×PBS分别将CMS5-VP1细胞稀释至1×10 7/mL和3×10 7/mL，CMS5细胞稀释至3×10 7/mL。
4)6-8周龄的BALB/c雌性小鼠随机分组，每组3只。分别背部皮下注射1×10 6或3×10 6的CMS5-VP1细胞，其中对照组背部皮下注射3×10 6的CMS5细胞。
5)接种肿瘤细胞后第4天开始每隔一天测量肿瘤大小。
6)部分肿瘤组织以200目铜网研磨，加入胰酶，37℃消化30分钟。
7)胰酶消化后以40目滤网过滤后，RPMI1640完全培养基洗涤细胞3次。
8)1000rpm收集细胞，免疫印迹检测VP1表达情况。
以统计学软件GraphPad Prism 6.0(GraphPad，La Jolla，CA，USA)对所有实验数据进行统计学分析，以平均值±标准平均值误差(SEM)表示。数据统计以t检验法比较各组之间的差异。nc(p＞0.05)表示无显著差异，*表示显著差异(p＜0.05)，**表示较显著差异(p＜0.01)，***表示非常显著差异(p＜0.001)，****表示极显著差异(p＜0.0001)。
根据免疫印迹鉴定结果，选择其中三株CMS5-VP1细胞E2、F5及B5G2开展成瘤实验。前期开展的CMS5成瘤实验采用的接种量为3×10 6细胞/小鼠。为了确定候选的三株CMS5-VP1细胞株的成瘤情况及CMS5-VP1细胞接种量，以CMS5细胞小鼠为阳性对照，考察CMS5-VP1细胞的成瘤性。
6-8周周龄BALB/c雌性小鼠随机分组，每组三只。分别皮下接种CMS5-VP1细胞，接种量分别为每只小鼠1×10 6或3×10 6，其中阳性对照CMS5细胞接种量为3×10 6。接种肿瘤后第4天开始，每两天测量肿瘤的大小。当肿瘤生长到2000mm 3时对小鼠实施安乐死，取肿瘤组织进行VP1表达量检测。
根据肿瘤的体积的计算公式(Tumor volume＝L×W 2/2(其中L为长度，W为宽度))计算肿瘤的体积，以GraphPadPrism制作肿瘤生长曲线。
生长曲线如图1所示，接种CMS5-VP1/F5细胞的小鼠在接种后的第8天肿瘤开始消退，第10天后肿瘤完全消退。而接种1×10 6CMS5-VP1/B5G2细胞的小鼠肿瘤生长缓慢，在接种后的第16天肿瘤完全消退，接种1×10 6CMS5-VP1/B5G2细胞的小鼠肿瘤在第14天开始消退，第16天肿瘤几乎不可测，对CMS5-VP1/B5G2荷瘤鼠实施安乐死后，取肿瘤组织，剪碎后以胰酶消化半小时，以RPMI160完全培养基洗涤三次后收集到肿瘤细胞，取1×10 7肿瘤细胞以1mL细胞裂解液4℃裂解2小时后，13000rpm离心收集细胞上清，-20℃冻存。接种3×10 6CMS5-VP1/E2的荷瘤小鼠肿瘤生长趋势与对照组CMS5没有明显差异，第18天开始肿瘤生长快速生长，第20天时肿瘤体积＞2000mm3，而接种1×10 6CMS5-VP1/E2的荷瘤小鼠肿瘤生长相对缓慢，在接种后第20天肿瘤大小为1000，对荷瘤小鼠实施安乐死，部分肿瘤组织以胰酶消化后收集细胞。1×10 7肿瘤细胞以1mL细胞裂解液4℃裂解2小时后，13000rpm离心收集细胞上清，-20℃冻存。
为了进一步确定CMS5-VP1细胞的稳定性，分别取CMS5-VP1/B5G2及CMS5-VP1/E2细胞裂解液进行免疫印迹检测，检测结果显示，CMS5-VP1荷瘤小鼠的肿瘤组织中表达VP1蛋白。
基础实验例2 MCV-VP1_VP2假病毒构建，表达验证
1、质粒构建
质粒pcDNA3.1-VP2由南京金斯瑞完成基因合成，通过内切酶Kpn I/Xho I***至载体pcDNA3.1多克隆位点，并经测序确定***位点的正确性。取pVAX1-VP1/DH5α、pcDNA3.1-VP2/DH5α甘油菌于LB平板(含Amp或Kan等对应抗生素)上划线，37℃培养箱培养过夜。分别挑取pVAX1-VP1/DH5α、pcDNA3.1-VP2/DH5α单克隆菌落于5mL LB(含Amp或Kan等对应抗生素)液体培养基中，37℃摇床震荡培养过夜。过夜培养的pVAX1-VP1/DH5α、pcDNA3.1-VP2/DH5α菌液以质粒小抽试剂盒提取质粒。
以质粒pcDNA3.1-VP2为载体、pVAX1-VP1作为***片段，以内切酶Hind III/Xho I进行双酶切分别获得线性化的载体pcDNA3.1及目的基因VP1，酶切体系如下表5，37℃酶切反应4h。
表5 双酶切反应体系
试剂 体积(μL)
pcDNA3.1-VP2/pVAX1-VP1 10
Hind III 1
Xho I 1
10×M buffer 3
去离子水 15
总体积 30
酶切结束后，以1％琼脂糖凝胶电泳。琼脂糖凝胶电泳结果表明条带大小正确。紫外灯 照射下对目的条带切胶并以天根胶回收试剂盒回收DNA，分别取1μL回收产物以琼脂糖凝胶电泳检测，条带大小与理论大小一致。
根据检测结果，确定酶连接体系进行酶连接反应，反应体系如下表6。
表6 酶连接反应体系
试剂 体积(μL)
VP1 16
pcDNA3.1 1
T4连接酶 1
10×T4连接酶缓冲液 2
总体积 20
室温连接反应1h后，将酶连接产物加入50μL的DH5α感受态细胞中，冰浴30min，随后42℃水浴90s，取500μL LB液体培养基加至细胞中，37℃摇床100rpm震荡培养30min，培养结束后13000rpm离心，弃上清，以200μL LB液体培养基重悬菌体，涂布于LB平板(含Amp)，37℃培养箱培养过夜。
过夜培养的平板上随机挑取17个单克隆菌落以PCR preMix进行菌落PCR，PCR产物以琼脂糖凝胶电泳检测。挑取阳性克隆菌落于5mL LB液体培养基中，37℃摇床震荡培养过夜。
过夜培养的阳性克隆菌液分别取1mL送测序，测序引物为通用引物T7-F/BGH-R。剩余菌液加入15％甘油，-20℃冻存。序列比对结果显示，pcDNA3.1-VP1序列正确。
分别取pcDNA3.1-VP1、pcDNA3.1-VP2、pcDNA3.1-EGFP甘油菌于100mL LB液体培养基中，37℃摇床震荡培养过夜后，8000×g离心30min，收集菌体。
以QIAGEN中抽质粒试剂盒提取质粒，UV260/280测定质粒浓度，质粒浓度如下表7。
表7 质粒浓度表
质粒 浓度(mg/μL)
pcDNA3.1-VP1 1.07
pcDNA3.1-VP2 1.05
pcDNA3.1-EGFP 1.14
以Hind III/Xho I双酶切鉴定质粒，酶切鉴定结果表明，双酶切得到的条带大小正确，说明质粒正确。
2、MCV假病毒包装
1)MCV假病毒包装条件摸索
复苏293FT细胞，细胞稳定培养后，以胰酶消化细胞，1000×g离心5min收集细胞，以1mL DMEM完全培养基重悬细胞，取1μL细胞悬液稀释100倍后以台盼蓝染色，显微镜下计细胞数；
根据细胞计数结果以DMEM完全培养基将细胞稀释至1×10 6/mL；
细胞稀释液加入6孔板中，每孔2mL，5％CO 2培养箱中37℃培养过夜；
将pcDNA3.1-VP1、pcDNA3.1-VP2、pcDNA3.1-EGFP质粒按下表8比例混匀，参照Lipofectamine 2000操作手册进行共转染；
表8 共转染比例表
组别 pcDNA3.1-VP1(μg) pcDNA3.1-VP2(μg) pcDNA3.1-EGFP(μg)
1 1.2 1.2 0.6
2 1.8 0.3 0.9
3 1.5 0.75 0.75
4 2.57 0.42 0
5 0 1.5 1.5
6 2 0 1
48h后荧光显微镜下观察；
参考假病毒操作手册，以胰酶消化细胞后，以PBS洗涤并收集细胞，以细胞裂解液(Brij58)重悬细胞，加入0.1％的Benzonase，CO 2培养箱中37℃消化过夜以促进MCV假病毒包装及成熟；
CO 2培养箱中37℃培养的293FT细胞计数后，稀释至1.5×10 6/mL，取一96孔细胞培养板，加入细胞稀释液，每孔100μL；
细胞裂解液5000×g离心10min，收集上清，即为假病毒液；分别取各组假病毒以DMEM完全培养基稀释，起始稀释梯度为1:1000，10倍比例连续稀释7个梯度；
将各梯度假病毒稀释液加入过夜培养的293FT细胞96孔板中，每孔100μL假病毒稀释液，每个梯度2个复孔，CO 2培养箱中37℃培养72h。
荧光显微镜下观察各孔中绿色荧光蛋白EGFP的表达情况。
结果显示，制备的假病毒不具备感染293FT细胞的能力。共转染过程中荧光蛋白表达强度高，为了确定结构蛋白是否表达，各组假病毒悬液各取20μL，以抗VP1小鼠血清为一抗进行免疫印迹检测。
从免疫印迹结果看，除了5号组外各组均有VP1的表达。为了判断是否由于EGFP表达过强导致包装质粒效率太低导致未能检测到293FT的感染，因而采用其他表达强度相对弱的Ds-Red作为报道基因进行假病毒包装。共转染比例如下表9，共转染48h后荧光显微镜观察荧光蛋白的表达情况。
表9 共转染质粒比例
组别 pcDNA3.1-VP1(μg) pcDNA3.1-VP2(μg) pcDNA3.1-EGFP(μg)/Ds-Red
1 1.9 1.9 0.2
2 3.25 0.55 0.2
3 2.85 0.95 0.2
4 3.8 0 0.2
5 0 3.8 0.2
6 3 1 0
根据假病毒操作手册，获得MCV-EGFP、MCV-Ds-Red假病毒液，CO 2培养箱中37℃培养的293FT细胞计数后，稀释至1.5×10 6/mL，取一96孔细胞培养板，加入细胞稀释液，每孔100μL；
细胞裂解液5000×g离心10min，收集上清，即为假病毒悬液；分别取各组假病毒以DMEM完全培养基稀释，起始稀释梯度为1:1000，10倍比例连续稀释7个梯度；
将各梯度假病毒稀释液加入过夜培养的293FT细胞96孔板中，每孔100μL假病毒稀释液，每个梯度2个复孔，CO 2培养箱中37℃培养72h。
荧光显微镜下观察各孔中绿色荧光蛋白EGFP或红色荧光蛋白Ds-Red的表达情况。
结果表明，即便选择弱表达Ds-Red作为报道基因包装的假病毒依然不具备感染293FT细胞的能力。
荧光蛋白质粒Zs-Green作为报道基因在1×10 7细胞中包装的假病毒不具备感染293FT的能力。
同时采用pE2-GFP、pRwB作为报道基因以1×10 7细胞包装假病毒。
根据操作手册完成过假病毒包装及成熟后取假病毒悬液，1：1000为起始稀释度，10倍连续稀释7个梯度，于96孔板中开展感染293FT细胞实验，观察各孔中荧光强度。RwB作为报道基因的假病毒感染293FT的能力微弱。
实施例1 一种针对默克尔细胞癌的DNA疫苗
将SEQ ID NO.1所示的核苷酸序列克隆至pVAX1载体上，克隆使用的酶切位点为XbaI、HindIII。
克隆的具体步骤为本领域常规方法，在此不做赘述。克隆方法包括PCR扩增、酶切、连接及其相关步骤。
经鉴定成功将SEQ ID NO.1所示的核苷酸序列克隆至pVAX1载体上获得的重组载体依据本领域常规的命名惯例，命名为pVAX1-MCV-VP1。
鉴定的方法为本领域常规方法，在此不做赘述。例如双酶切鉴定或测序鉴定。经过XbaI、HindIII双酶切之后，能观察到一条大约为1.3kb的DNA条带。
pVAX1-MCV-VP1使用本领域常规的试剂盒进行扩增，例如天根生化科技(北京)有限公司的无内毒素质粒大提试剂盒(货号DP117)。
本领域技术人员应当理解的是，无论是经克隆直接得到的pVAX1-MCV-VP1还是经过试剂盒扩增得到的pVAX-MCV-VP1扩增子，均为本实施例制备的针对默克尔细胞癌的DNA疫苗。
实施例2 一种针对默克尔细胞癌的DNA疫苗
本实施例与实施例1的唯一区别在于，使用SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的核苷酸序列替换SEQ ID NO.1所示的核苷酸序列。
经鉴定成功将SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的核苷酸序列克隆至pVAX载体上获得的重组载体依据本领域常规的命名惯例，命名为pVAX-MCV-VP1-1、pVAX-MCV-VP1-2、pVAX-MCV-VP1-3。
实施例3 一种针对默克尔细胞癌的蛋白疫苗
1、制备方法：
(1)克隆：将SEQ ID NO.3所示的核苷酸序列，在其C末端连接一个6×His标签，通过内切酶XhoI与NcoI克隆至pET28a载体上，得到具有His标签的pET28a-VP1/Rosetta细胞(由)，将其置于-80℃冰箱保存。克隆使用的酶切位点为NcoI和XhoI。克隆的具体步骤在此不做赘述。所述的克隆方法包括PCR扩增、酶切、连接及其相关步骤。经鉴定成功将SEQ ID NO.3所示的核苷酸序列克隆至pET28a载体上获得的重组载体依据本领域常规的命名惯例，命名为pET28a-VP1。
(2)蛋白表达：表达可采用任何能够实现原核***蛋白表达的表达***。例如BL21或者Rosetta细胞表达，具体表达步骤参照BL21或者Rosetta细胞的使用手册进行，在此不做赘述。将pET28a-VP1转化至Rosetta细胞中，得到pET28a-VP1/Rosetta细胞，再将pET28a-VP1/Rosetta细胞的单克隆接种至相应抗性的LB培养基中，在37℃下扩大培养至OD 600＝0.6-0.8；IPTG诱导培养后，诱导条件为：18℃，1mM IPTG的诱导条件；离心收集菌体；超声破碎菌体；离心收集上清。
(3)蛋白纯化：使用AKTA AVANT纯化***以及镍柱(QIAGEN)对步骤(2)最终得到的上清进行纯化，具体纯化方法参照纯化***以及镍柱的产品使用说明。漂洗液体系为PBS+0.5M NaCl+100mM咪唑，pH＝7，清洗与镍柱非特异性结合的杂蛋白；洗脱液体系为PBS+0.5M NaCl+500mM咪唑，pH＝7，洗脱目的蛋白，得到含有目的蛋白的洗脱液。
(4)换液及体外组装：将步骤(3)得到的含有目的蛋白的洗脱液使用透析袋进行透析换液，透析条件为：
第一步透析条件：4℃进行，每隔6小时更换一次透析液，每次2L。透析外液依次为：PBS+0.5M NaCl、PBS+0.4M NaCl、PBS+0.3M NaCl、PBS+0.2M NaCl、PBS+0.1M NaCl、PBS；本步透析的目的为去除洗脱液中的多余盐组分；经PBS透析后，得到含有目的蛋白的去盐洗脱液；
第二步透析条件：将第一步透析后得到的含有目的蛋白的去盐洗脱液先浓缩，后稀释至蛋白浓度为0.1mg/mL，于0.5M硫酸铵、20mM Tris-base+5％甘油、1mM氯化钙、pH＝7条件下，在室温透析15小时。随后在4℃，PBS(pH＝7)中透析24小时。即得重组蛋白，重组蛋白经常规浓缩方法浓缩后(例如PEG8000粉末浓缩)，得到针对默克尔细胞癌的蛋白疫苗VP1。
2、验证实验：
针对本实施例制备的蛋白疫苗VP1进行性能检测，结果如图2所示。其中，A为不同诱导条件下的可溶表达情况：泳道1-18℃，IPTG 0mM；泳道2-18℃，IPTG 0.1mM；泳道3-18℃，IPTG 0.5mM；泳道4-18℃，IPTG 1mM；泳道5-18℃，IPTG 2mM；泳道6-30℃，IPTG 0mM；泳道7-30℃，IPTG 0.1mM；泳道8-30℃，IPTG 0.5mM；泳道9-30℃，IPTG 1mM；泳道10-30℃，IPTG 2mM；泳道a-上清蛋白；泳道b-包涵体蛋白质；泳道m-蛋白marker；B为镍柱纯化过程中的电泳图：泳道a-菌体蛋白；泳道b-上清液蛋白质；泳道c-包涵体蛋白质；泳道m-蛋白marker；泳道100mM-用100mM咪唑洗脱的组分；C为Western-Blot分析，抗体为抗His抗体：泳道1-OVA；泳道2-诱导后的重组蛋白；泳道3-纯化后的重组蛋白；D/E为Western-Blot分析，抗体为DNA疫苗免疫后小鼠血清；F为组装后蛋白疫苗VP1的电镜观察。
为了确定蛋白表达的最佳条件，采用不同的温度和IPTG浓度，并观察到在18℃，1mM IPTG的诱导条件下能够获得最高水平的可溶性VP1的表达(图2中的A)。在获得可溶性蛋白后，将其通过镍柱纯化，并用SDS-PAGE以及Western-Blot(anti-His的抗体或DNA疫苗免疫后的小鼠血清)对其纯度以及是否是目的蛋白进行验证(图2中的B/C/D)。
虽然MCV-VP1可以在真核***中自组装成VLP，通常MCV的包膜蛋白VP2也会参与到该过程中，但尚未报道VP1在原核***中也能自组装成VLP，尤其是在没有VP2参与的情况下。由于VLP的组装可能在很大程度上受外部条件的影响，主要包括pH，钠和钙离子的强度等因素，因此在探索VLP在原核***体外组装的过程中，采用了不同的外部条件。SDS-PAGE的结果显示，当用β-巯基乙醇(β-ME)处理样品后VP1蛋白约为47kD，但在没有β-ME处理的情况下却超过了250kD(图2中的D)，预示着也许通过自组装，形成了由72个VP1五聚体组成的VLP。接下来，将组装后的蛋白用蔗糖密度梯度离心，并用β-ME或不用β-ME进行SDS-PAGE和Western Blot，进一步证实该VLP形成(图2中的E)。最后，通过透射电子显微镜进一步检查，观察到了不同大小的VLP颗粒(图2中的F)。
总之，结果表明，具有SEQ ID NO.3所示的核苷酸序列的MCV-VP1可以在大肠杆菌中以可溶性蛋白的形式表达，并通过体外自组装形成不同大小的颗粒。
部分实验方法及条件举例：
透射电镜实验：将上述最终透析后获得的重组蛋白(即蛋白疫苗VP1)通过30％-60％的蔗糖密度梯度离心；之后吸取了不同组分层间的样品，以验证目的蛋白离心后的位置。将通过蔗糖密度梯度离心的目的蛋白用磷钨酸染液染色，通过透射电镜，在电压80kV的条件下观察是否有病毒样颗粒存在。
SDS-PAGE：胶浓度12％，电泳条件为，浓缩胶80V电压约30分钟，分离胶120V电压约1小时。
Western-Blot(免疫印迹)：电泳结束后，若不需要做免疫印迹，则用G250染色并脱色后，拍照。若需要做免疫印迹，则将蛋白转移到PVDF膜上；用含有脱脂奶粉的TBST(含有吐温20的Tris缓冲液)将PVDF膜依封闭一小时，随后一抗孵育一小时，二抗(山羊抗小鼠IgG-HRP，稀释度1：4000)孵育一小时，一抗二抗均用含2％脱脂奶粉的TBST稀释；用Pro-Light HRP显色液(天根)对PVDF膜进行显色。
实施例4 一种针对默克尔细胞癌的蛋白疫苗
本实施例与实施例3的唯一区别在于，使用SEQ ID NO.1所示的核苷酸序列替换SEQ ID NO.3所示的核苷酸序列。
实施例5 pET28a-VP1/Rosetta发酵工艺的建立
委托南京金斯瑞公司进行基因合成及质粒构建工作。
为了获得大量的VP1重组蛋白，以10L发酵罐生产。具体包括以下步骤：
1、种子培养
1)甘油菌划线培养：-80℃冰箱中取出实施例3制得的具有His标签的pET28a-VP1/Rosetta甘油菌，室温解冻。在超净台中，用接种针蘸取甘油菌液在含有100μg/mL硫酸卡钠霉素(Kan)的抗生素LB平板上划线，37℃恒温培养箱中培养过夜。
2)一级种子培养：在超净工作台中，从平板上一个单克隆菌落，接种于20mL LB液体培养基(含100μg/mL Kan)中，培养温度37℃，摇床转速200rpm，培养16小时左右。
3)二级种子接种：培养好的一级种子取10mL加入500mL培养基(含100μg/mL Kan)中，培养温度37℃，摇床转速200rpm，培养3小时左右，至OD600在1-2之间，待上罐接种。
2、发酵罐发酵
1)检查发酵罐各部件状态后校正pH电极、并安装号传感器、管路、街头及滤器等配件。
2)发酵罐内配制4L罐上培养基，121℃灭菌20分钟，灭菌过程中将溶氧调零。罐灭菌后冷却至37℃待上罐接种。
3、补料和补料管的准备
配制总体积为2.5L的补料培养基，装于补料瓶中，与各补料管和补料头，115℃灭菌20分钟。
配制消泡剂200mL，121℃灭菌20分钟。
一个无菌的补料瓶倒入氨水待用。
4、发酵罐接种前准备
1)从接种口加入Kan、微量元素和葡萄糖。
2)调试并设置好发酵参数。以氨水将培养基pH调至7.0。校正溶氧电极100值，并设定工艺参数：40％的DO(溶氧浓度)；起始转速200rpm；200L/小时的空气流量；起始pH：7.0；温度：37℃。
5、接种
将500mL培养好的二级种子液(OD 600为1-2)接入发酵罐，记录接种时间、接种时菌种OD 600值。
6、培养阶段
1)pH的控制：自动补加氨水，控制在pH7.0左右；
2)DO控制：大于等于40％，设定40％，低于此值增加转速和气体控制；
3)转速的控制：根据溶氧调整，满足溶氧需求，最高600转；
4)泡沫控制：间歇补加消泡剂(补加原则为尽量少加消泡剂，控制液面不喷罐即可)。
5)补加的控制：视溶氧、pH情况加补。
6)过程检测：每小时测OD600值并记录发酵情况。
7、诱导阶段
在OD 600达到30左右时加入1mM IPTG进行诱导，温度降至30℃，诱导4小时。
8、收罐
诱导4h后收罐，发酵液离心，8000rpm离心30分钟。弃上清，沉淀用磷酸钠(pH7.0)缓冲液洗涤2次，收集后待用。
取少量菌体，以SDS-PAGE及免疫印迹鉴定表达情况。
实验结果：
本研究使用的MCV疫苗抗原VP1采用大肠杆菌原核表达***，以IPTG诱导表达。密码子优化后的VP1通过分子克隆技术构建至原核表达载体pET28a上，质粒命名为pET28a-VP1，测序及酶切鉴定质粒的正确性。序列正确的pET28a-VP1质粒采用化学法转化至大肠杆菌表达菌株Rosetta感受态细胞中，命名为pET28a-VP1/Rosetta，表达菌株1:100接种于含100μg/mL Kan的LB液体培养基中，37℃振荡培养过夜后，将过夜培养的菌液分装于1.5mL无菌离心管中，每管750μL菌液与250μL 60％无菌甘油混匀，-20℃，即为MCV-VP1发酵种子。
pET28a-VP1/Rosetta扩大培养后接种发酵罐中37℃发酵培养10.5h后OD 600为24.9，将温度降到30℃，以0.5mM IPTG诱导VP1蛋白表达3h，OD 600达35.2，放罐收集发酵液，8000g离心30分钟后收集菌体称重，获得400g湿菌。
0.1g菌体重悬于1.5mL破菌缓冲液中，高压均质机破菌后离心，分别收集破菌上清及沉淀，沉淀以1.5mL裂解液重悬，裂解过夜。裂解液离心后分别收集上清、沉淀。沉淀以1.5mL裂解液重悬后，分别取50μL破菌上清、破菌沉淀及裂解上清，加入10μL 6×loading buffer，煮沸10分钟，以12％SDS-PAGE电泳(100V 30分钟，150V 60分钟)，考马斯亮蓝染色4h，脱色液脱色过夜。同时以抗His tag鼠多抗为一抗、HRP标记的羊抗鼠IgG为二抗进行免疫印迹检测。
SDS-PAGE电泳结果表明，目的蛋白VP1大小为46kDa，泳道2、3在45kDa左右有明显的条带，从SDS-PAGE结果可确定发酵罐30℃诱导表达的VP1主要形成包涵体。以抗His tag鼠多抗为一抗的免疫印迹结果进一步证实目的蛋白VP1主要形成包涵体。
实施例6 重组蛋白VP1的纯化
采用镍离子亲和层析柱纯化VP1重组蛋白。
1、破菌
取10g实施例5中制得的MCV-VP1表达菌体，以150mL破菌缓冲液(0.1M NaH 2PO 4、20mM Tris、0.3M NaCl，pH7.0)重悬菌体。以高压均质机均质30分钟，8000×g离心30分钟，收集菌体。
2、溶菌
以150mL裂解液(0.1M NaH 2PO 4、20mM Tris、0.3M NaCl、8M尿素，pH7.0)重悬菌体，调节pH至7.0，4℃裂解过夜。
3、离子亲和层析纯化
1)8000×g离心15分钟，收集上清。
2)取20mL偶联了镍离子的Ni-NTA于层析柱中，连接至AKATA。
3)以5倍柱体积的去离子水洗涤后，以5倍柱体积的平衡缓冲液(0.1M NaH 2PO 4、20mM Tris、0.3M NaCl、8M尿素，pH7.0)平衡Ni-NTA。
4)裂解上清以2mL/分钟的速度上样，同时收集流穿液。
5)以5倍柱体积的洗涤缓冲液(0.1M NaH 2PO 4、20mM Tris、0.3M NaCl、8M尿素、20mM Imidazole，pH7.0)冲洗柱子，流速为5mL/分钟，同时收集洗脱液。
6)以洗脱液(0.1M NaH 2PO 4、20mM Tris、0.3M NaCl、8M尿素、500mMImidazole， pH7.0)洗脱目的蛋白，流速为2mL/分钟，分步收集洗脱下来的目的蛋白。
7)以0.5M NaOH冲洗柱子，流速为10mL/分钟。
8)以去离子水洗涤柱子，流速为10mL/mL。
9)洗脱得到的目的蛋白通过SDS-PAGE及免疫印迹检测目的蛋白。
实验结果：
包涵体表达的VP1以破菌缓冲液重悬后，以高压均质机机械法破菌，破菌沉淀以含有8M尿素的裂解液裂解过夜后，离心取得的上清在GE公司AKATA prime机器上通过镍离子亲和层析柱纯化VP1。
SDS-PAGE检测纯化后的蛋白，结果如图3所示，其中，M表示蛋白质预染Marker(天根生化科技，MP206)；1为破菌上清；2为裂解上清；3为流穿液；4为20mM咪唑洗涤液；5为100mM洗涤液；6为250mM洗脱液；7为500mM洗脱液；8为0.5M氢氧化钠洗涤液。从图3可以看出，250mM和500mM咪唑洗脱下来的目的蛋白为单一条带。
将图3中6、7号样品合并后，以透析袋透析逐步去除尿素，在含2M尿素的缓冲液中透析过夜后，在不同透析缓冲液中透析过夜以完全去除尿素以复性VP1。复性的VP1会自发地组装成以五聚体为亚单位的蛋白颗粒。参考文献报道的SV40-VP1或MCV-VP1组装条件，采用了以下不同的缓冲体系进行自组装。
Buffer1:100mM NaH2PO4,20mM Tris,pH7.2。
Buffer2:100mM NaH2PO4,20mM Tris,2M(NH4)2SO4,2mM CaCl2,pH7.2。
Buffer3:100mM NaH2PO4,20mM Tris,1M NaCl,pH7.2。
Buffer4:100mM NaH2PO4,20mM Tris,150mM NaCl,2mM CaCl2,pH7.2。
Buffer5:20mM MOPS,0.3M NaCl,pH7.0。
实施例7 MCV-VP1重组装工艺研究
本实施例在实施例6的基础上进行进一步研究。
1、重组蛋白VP1的复性
共价二硫键在蛋白形成高级结构中发挥着重要作用。还原性试剂如DTT和高浓度的尿素可分别破坏错误共价二硫键和离子键从而打开蛋白的高级结构，使蛋白以单体分子的形式存在，此过程为蛋白变性。当还原性试剂如DTT和尿素浓度逐渐降低过程中，二硫键逐步恢复，蛋白可自发地折叠形成高级结构，该过程成为蛋白复性。变性条件下纯化得到的VP1通过透析逐步去除蛋白溶液中的尿素以复性，具体方法如下：
1)含8M尿素的VP1溶液装入透析袋中，于溶液1(0.1M NaH 2PO 4、20mM Tris、0.3M NaCl、6M尿素，pH7.0)中透析过夜。
2)随后于溶液2(0.1M NaH 2PO 4、20mM Tris、0.3M NaCl、4M尿素，pH7.0)中透析过夜。
3)将透析过夜的VP1蛋白于溶液3(0.1M NaH 2PO 4、20mM Tris、0.3M NaCl、2M尿素，pH7.0)中透析过夜。
4)自组装工艺：在不同的组装缓冲液中透析过夜以去除VP1蛋白中的尿素，此时VP1完全复性，会自发地组装成VP1结构蛋白。
5)以Nu-PAGE检测VP1是否形成五聚体。以纳米粒径仪检测MCV-VP1颗粒大小。
2、MCV-VP1重组装
以包涵体形式表达的重组蛋白须在变形条件下纯化，蛋白复性过程中易发生错误折叠而导致蛋白构象发生改变而影响蛋白地活性。解组装、重组装工艺如下：
1)自组装的VP1加入10mM的DTT及10mM的EGTA，水平摇床上低速混匀2小时，此过程为解组装过程。
2)解组装的VP1蛋白装入透析袋中，在五种不同的重组装条件下进行重组装，具体为：
Buffer1:100mM NaH2PO4,20mM Tris,pH7.2。
Buffer2:100mM NaH2PO4,20mM Tris,2M(NH4)2SO4,2mM CaCl2,pH7.2。
Buffer3:100mM NaH2PO4,20mM Tris,1M NaCl,pH7.2。
Buffer4:100mM NaH2PO4,20mM Tris,150mM NaCl,2mM CaCl2,pH7.2。
Buffer5:20mM MOPS,0.3M NaCl,pH7.0。
3)将重组装(也称为自组装)的重组VP1命名为(rVP1)，再将rVP1以0.22um针头式滤器过滤除菌后，以Nu-PAGE检测rVP1的5具体结构，并以纳米粒径仪检测rVP1颗粒大小。
结果：
野生型MCV是以VP1五聚体为基本单位组成的一个具有72面体的病毒颗粒。有文献报道，重组表达的多瘤病毒衣壳蛋白VP1还会以多聚体的结构存在。通过Nu-PAGE来检测不同组装工艺下形成的VP1结构蛋白是否是以五聚体为基本单位。Nu-PAGE电泳检测结果如图4所示，图4中的1-5分别表示在上述不同重组装条件下组装VP1，M，蛋白质非预染Marker，结果表明，在不同组装条件下组装VP1(rVP1)形成五聚体外，还以多聚体的形式存在。
通过马尔文纳米粒径仪检测了自组装VP1(rVP1)颗粒的大小，结果表明自组装的VP1(rVP1)结构蛋白形成了大小不一的颗粒。为了获得稳定的蛋白颗粒，接下来以DTT及EGTA对自组装的VP1结构蛋白进行解组装-重组装工艺研究，希望通过重组装工艺获得更加接近野生型MCV颗粒大小的重组蛋白颗粒。
3、MCV-VP1重组装(rVP1)
为了获得颗粒稳定的结构蛋白，自组装的VP1(rVP1)结构蛋白以10mM DTT、10mM EGTA解组装后，在不同的重组装条件下开展VP1重组装工艺探索，最终确定了氧化还原剂(GSSG-GSH)条件的重组装工艺。
VP1解组装后，在重组装缓冲液(50mM Tris,0.8M(NH 4) 2SO 4,0.2M NaCl,0.5mM GSH,4.5mM GSSG,2mM CaCl 2,pH6.4)透析48小时，随后在缓冲液(50mM Tris,0.2M NaCl,pH7.0)中透析过夜以去除蛋白溶液的(NH 4) 2SO 4及CaCl 2。通过这些工艺获得了重组装VP1(rVP1)。
分别以SDS-PAGE、免疫印迹检测重组装VP1(rVP1)、非还原电泳检测VP1五聚体结构，rVP1变性条件下呈现唯一条带。以Themofisher公司的Nu-PAGE检测，VP1单体分子量为46kDa，五聚体分子量大概在250kDa左右。因此，可以认为250kDa处的条带是VP1五聚体结构m，从该结果认为重组装工艺下获得的rVP1主要以五聚体为基本单位组成蛋白颗。
同时，以纳米粒径仪测定rVP1及低温诱导表达的可溶性VP1(sVP1)的颗粒大小，结果相比VP1自组装颗粒，rVP1颗粒更为均一，粒径为115.3nm。而sVP1的颗粒远远大于rVP1，这一结果说明重组装过程中氧化-还原剂的存在有助于重组装过程中结构的稳定性。
随后取5g湿菌以该纯化工艺获得了150mg VP1重组蛋白，本研究在10L发酵罐中以8L培养基进行发酵获得了400g的湿菌，因此，VP1重组蛋白的表达量高达1.5g/L。
实施例8 蛋白疫苗免疫原性的验证
为了验证实施例3制备的蛋白疫苗VP1的免疫原性，用不同剂量的蛋白疫苗VP1(1.1μg/只、3.3μg/只、10μg/只、20μg/只)每两周一次共两次免疫小鼠，采用的免疫方法为铝佐剂吸附以及肌肉注射。将注射蛋白疫苗第一天计为0天。
同时为了筛选最佳的免疫剂量，在终免后7天检测了小鼠特异性MCV-VP1抗体滴度以及DTH(迟发型超敏反应)之后的足垫厚度(图5中的B/C/D)。结果表明：在不同剂量的蛋白疫苗免疫组中均能检测到抗MCV-VP1特异性抗体滴度，但在10μg组中观察到了最高的抗体滴度(图5中的B)。虽然从DTH的结果来看，3.3μg组的DTH水平最高，但是10μg组的DTH水平也相对较高(图5中的C/D)。因此，综合特异性抗体水平及DTH的结果来看，最后选择了10μg作为蛋白疫苗的最佳剂量，并在下面的实验中采用该剂量(图中VP1指蛋白疫苗免疫组)。
为了进一步评估蛋白疫苗引发的抗原特异性细胞应答，预测并合成了VP1特异性的肽池MHC-I/MHC-II表位肽(MCV-VP1peptide)，并用该肽池刺激了从蛋白疫苗免疫后小鼠中分离到的脾细胞。结果表明，蛋白疫苗免疫组(MCV-VP1)中CD8 +T细胞的IFN-γ和TNF-α水平显著高于PBS免疫组(Vehicle)，而CD4 +T细胞的IL-4、CD8 +T细胞的Granzyme B的水平也呈现出相同的变化趋势(图5中的E)。
本实验例中使用的小鼠为6-8周的BALB/c和C57BL/6小鼠，均从杰斯捷公司购买。DNA疫苗通过肌肉注射联合电脉冲免疫，蛋白疫苗通过蛋白加铝佐剂肌肉注射免疫。部分实验方法或条件列举如下：
迟发型超敏反应(DTH)：疫苗免疫小鼠，终免后7天，向小鼠免疫侧(右侧)后足垫注射10μL抗原蛋白(10μg)，免疫对侧(左侧)后足垫注射10μL磷酸盐缓冲液。DTH后24，48小时用游标卡尺测量小鼠两只后足垫厚度，相同部位测量三次取平均值。DTH值(mm)＝右侧足垫厚度-左侧足垫厚度。
酶联免疫吸附测定：第21天采集小鼠血液样品，用ELISA法测定血清的特异性抗体滴度。当需要半定量检测小鼠血清中的抗体浓度时，在常规ELISA的基础上增加标准曲线，根据标准曲线的浓度来判定小鼠血清中抗体的浓度。
ELISA检测sVP1特异性抗体滴度方法：
完成免疫程序的BALB/c在初次免疫后的第28天眼底取血，4℃静置过夜。6000rpm离心10分钟，取血清。通过ELISA检测血清中抗VP1抗体水平，具体方法如下：
包被：rVP1以抗原包被液稀释至2μg/mL,以100μL/孔的抗原稀释液包被96孔ELISA板，4℃过夜。
封闭：包被过夜的96孔板甩去包被液，1×PBST洗涤一次。加入含5％脱脂奶粉的1×PBST，150μL/孔，37℃孵育1小时。
一抗稀释：取小鼠血清，以含2％脱脂奶粉的1×PBST按1:800稀释后，连续对倍稀释8个梯度。VP1兔多抗以含2％脱脂奶粉的1×PBST按1:800稀释后，连续对倍稀释8个梯度作为阳性对照。以Al(OH) 3组血清1:800稀释度作为阴性对照。
一抗孵育：封闭结束后，甩去封闭液，以1×PBST洗涤3次。甩干后，将稀释好的鼠血清加至96孔板中，100μL/孔，每个样品两个复孔。37℃孵育1小时。
二抗孵育：HRP标记的羊抗鼠IgG及HRP标记的羊抗兔IgG分别以含2％脱脂奶粉的1×PBST稀释1000倍。一抗孵育结束后，将一抗甩去，以1×PBST洗涤5次，甩干。100μL/孔加入稀释好的HRP标记的羊抗鼠IgG，其中阳性对照加入HRP标记的羊抗兔IgG。37℃孵育1小时。
显色：二抗孵育结束后，甩去二抗。以1×PBST洗涤5次，甩干。100μL/孔加入TMB显色液，37℃孵育5分钟。
终止：显色结束后，加入50μL的2M H 2SO 4。
读数：以酶标仪在波长450nm/620nm下读取光吸收值。
数据分析：以阴性对照组OD值的2.1倍作为cut off值，样品组OD值大于cut off时即为阳性，每个样品最后一个阳性孔的稀释度即为该样品的抗体滴度。
脾细胞特异性刺激：无菌环境中进行，最终免疫后7天的小鼠脱颈安乐死，取出脾脏，研磨成单细胞悬液；离心收获细胞，红细胞裂解液重悬后裂解含FBS的PBS终止裂解；过滤，PBS清洗后用含有anti-CD28(1：5000稀释)的1640完全培养基重悬细胞；对制备好的单细胞悬液计数，并用1×10 6个细胞/孔铺板；每只小鼠的样品共设置三个重复，包括未刺激组、VP1特异性多肽刺激组(MCV-VP1 Peptide)、佛波酯/离子霉素阳性刺激组(PMA+Ino)，VP1特异性多肽池如表10所示，多肽工作浓度为10μg/mL，PMA工作浓度为0.1μg/mL，Ino工作浓度为工作浓度1μg/mL。同时，混合物中应含有1：1000比例的 BFA(Brefeldin A)，以抑制细胞因子分泌到胞外；37℃，5％CO 2培养6hr；离心收集刺激完的细胞。流式细胞计数检测。
表10 VP1特异性多肽池
Figure PCTCN2021127386-appb-000005
流式细胞技术：死活染色：使用EF780-APC-Cy7抗体，常温摇床避光染色；细胞表面染色：用表面抗体染色液(CD3-FITC/CD4-BV421/CD8-Percp-Cy5.5)，染色步骤与细胞死活染色一致；胞内染色：使用了Foxp3/转录因子染色试剂盒(eBioscience)；流式细胞仪LSRFortssa(Becton)检测。
实施例9 DNA疫苗的免疫应答
蛋白疫苗主要激活机体的体液免疫反应，而细胞免疫反应较弱。因此，进一步对实施例1制备得到的针对默克尔细胞癌的DNA疫苗进行验证。
将实施例1得到的pVAX1-MCV-VP1(即DNA疫苗)通过用固定剂量的DNA疫苗以不同的时间间隔肌肉注射联合电脉冲免疫小鼠3次，发现在第0，7和14天免疫小鼠能够获得最高的DTH和特异性抗体应答(图6中的A、B)。为了确定最佳免疫剂量，选择了从12.5μg/只到100μg/只不同剂量的DNA疫苗，以间隔一周肌肉免疫，共免疫三次的策略免疫小鼠。确定了最佳免疫剂量为100μg。此外，pVAX1-MCV-VP1 DNA疫苗免疫后的小鼠，其脾脏细胞在经过特异性肽池刺激后，CD4 +T细胞的IFN-γ和CD4 +T细胞的TNF-α水平显著增加，而增加更为显著的是CD8 +T细胞的IFN-γ，达到了5倍左右(图6中的E、F)。以上结果表明pVAX1-MCV-VP1 DNA疫苗可以诱导机体产生强烈的VP1特异性细胞免疫应答。
DNA疫苗的免疫应答验证结果见图6。其中，A/B为最佳免疫策略研究：pVAX1-OVA-2-3为在第0，14，28天用pVAX1-OVA肌肉注射联合电脉冲免疫小鼠；pVAX1-OVA n 2-3为在第0，14，28天用pVAX1-OVA肌肉免疫小鼠，免疫时未电脉冲；pVAX1-MCV-VP1 2-2为在第0，14天用pVAX1-MCV-VP肌肉注射联合电脉冲免疫小鼠；pVAX1-MCV-VP1 1-3为在第0，7，14天用pVAX1-MCV-VP1肌肉注射联合电脉冲免疫小鼠；pVAX1-MCV-VP1 2-3为在第0，14，28天用pVAX1-MCV-VP1肌肉注射联合电脉冲免疫小鼠；pVAX1-MCV-VP1 n 2-3为在第0，14，28天用pVAX1-MCV-VP1肌肉注射联合电脉冲免疫小鼠，免疫时未电 脉冲。C/D为最佳免疫剂量摸索。E/F为脾细胞特异性体外刺激。
实施例10 联合免疫反应
由于针对默克尔细胞癌的蛋白疫苗可以诱导更高水平的特异性抗体反应，而DNA疫苗则能诱导更高的细胞免疫反应，因此，本实施例验证了将实施例1制备的DNA疫苗以及实施例3制备的蛋白疫苗结合的情况下能否同时诱导较高水平的细胞和体液免疫反应。
为了达到这个目的，在第0、14、28天对小鼠进行免疫，无论是DNA疫苗还是蛋白疫苗，都采用实施例8和实施例9中摸索到的相应最佳免疫剂量。
终免后7天的DTH结果如图7中的A所示，D代表一针DNA疫苗，P代表一针蛋白疫苗。
该结果表明一针DNA疫苗加两针蛋白疫苗(D-P-P)的免疫策略能够诱导出最高水平的DTH反应，其次依次为D-D-P，D-D-D和P-P-P。另一方面，对于VP1特异性抗体滴度水平，最佳的免疫策略是D-P-P，然后是P-P-P，D-D-P和D-D-D(图7中的B)。从不同免疫策略间的比较来看，一针DNA疫苗加两针蛋白疫苗(DPP)的免疫策略能够实现更高水平的抗VP1抗体以及DTH反应，这表明该免疫策略能够同时引起体液和细胞免疫反应。
实施例11 MCV-VP1治疗性疫苗治疗效果研究
细胞因子rhGM-CSF购自华北制药金坦生物制药有限公司。
重组鼠源IFN-α2b(rmIFN-α2b)购自北京义翘神州(50525-MNAY)。
CpG1826由上海杰瑞生物合成。
本研究以rhGM-CSF和IFN-α2b(rmIFN-α2b)或者CpG、R848或MPL联合Al(OH) 3为佐剂制备了MCV治疗性疫苗，并开展MCV治疗性疫苗的抗肿瘤作用研究。
R848购自invivogen公司(tlrl-r848)。
实验中流式细胞检测中用到的荧光标记抗体Biolegend公司或BD公司。
嘌呤霉素(Puromycin)购自上海翊圣生物科技有限公司。
细胞培养用RPMI1640、胎牛血清(FBS)、胰酶及青霉素/链霉素双抗购自BI公司。
血球计数板、台盼蓝、购自Thermo fisher公司。
实验中所使用的6-8周周龄BALB/c小鼠雌性小鼠，购自上海斯莱克实验动物有限责任公司，饲养在复旦大学实验动物科学部SPF清洁级鼠房，所有动物实验操作均在动物保护指导条例指导下进行。
1、MCV治疗性疫苗药效评价
1)CMS5-VP1细胞培养
前期实验通过脂质体转染法将慢病毒质粒pcDH-VP1转染至BALB/c背景的肿瘤细胞CMS5中，在5μg/mL的Puromycin药物压力下筛选到了稳定表达VP1的细胞株CMS5-VP1/E2。以BALB/c小鼠开展的成瘤实验确定了肿瘤模型的构建方式为皮下注射1×10 6的细胞。(见基础实施例1)
为了开展MCV治疗性疫苗的药效评价，将冻存于-80℃的CMS5-VP1/E2细胞进行复苏。具体方法为：CMS5-VP1/E2细胞冻存管于37℃水浴解冻后，以RPMI1640完全培养基洗涤2次后，1500rpm离心5分钟，收集细胞。以含有5μg/mL Puromycin的RPMI1640完全培养基重悬细胞后，于10cm细胞培养皿中5％CO 2培养箱37℃培养过夜。
待复苏的细胞长至占培养皿80％时，胰酶消化后，1:2的比例传代于2个10cm细胞培养皿中。当细胞长满至占培养皿80％时，胰酶消化后1:2的比例传代于T75 Flask中。随后传代于4个T175Flask中，如此传代至第6代。
胰酶消化后，以PBS洗涤细胞2次，1500rpm离心5分钟，收集细胞。PBS重悬细胞后，以40μm孔径的网筛过滤细胞，制成单细胞悬液。取部分细胞稀释后，台盼蓝染色、显微镜下以细胞计数板进行计数。以PBS将单细胞悬液稀释至1×10 7/mL，置于4℃冰盒中备 用。
2)肿瘤模型建立
6-8周周龄的BALB/c雌性小鼠随机分笼，每组5只。小鼠背部皮下接种CMS5-VP1细胞悬液，接种量为1×10 6/100μL。
3)MCV治疗性疫苗制备及接种
将实施例7中制得的重组装蛋白rVP1(1.8mg/mL)以缓冲液稀释后，1:1的比例与10mg/mL的Al(OH) 3混匀后，4℃摇床中低速震荡过夜以利于抗原均匀地吸附于Al(OH) 3佐剂，即为MCV重组蛋白疫苗半成品。
CMS5-VP1细胞接种后第5天，MCV重组蛋白疫苗半成品与佐剂GM-CSF/IFN-α、CpG、R848或MPL按一定比例混匀即为MCV治疗性疫苗，4℃摇床中低速震荡2h。分别于小鼠的后肢肌肉注射MCV治疗性疫苗。
分别于肿瘤接种后的5、12、19天肌肉注射MCV治疗性疫苗组及其余各组，其中各组分别为：
Vac/G+I(10μg rVP1/10μg GM-CSF/1μg IFN-α/500μg Al(OH) 3)；
Vac/C(10μg rVP1/10μg CpG/500μg Al(OH) 3)；
Vac/R(10μg rVP1/10μg R848/500μg Al(OH) 3)；
Vac/M(10μg rVP1/25μg MPL/500μg Al(OH) 3)；
Vac(10μg rVP1/500μg Al(OH) 3)；
C/A(10μgCpG/500μg Al(OH)3)；
R/A(10μg R848/500μg Al(OH)3)；
Al(OH)3(500μg Al(OH)3)；
Vehicle(100μl PBS)。
4)肿瘤生长曲线监测
CMS5-VP1细胞接种后第5天开始测量肿瘤的大小，每两天测量一次，并计算肿瘤的体积。以GraphPad Prism软件绘制肿瘤生长曲线图。肿瘤体积的计算公式为：Tumor volume(mm 3)＝L×W 2/2。
实验结果：
1、MCV治疗性疫苗效果评价
为了评价MCV治疗性疫苗的治疗效果，本研究通过构建了一个稳定表达VP1的肿瘤细胞系CMS5-VP1以建立肿瘤模型来评价MCV治疗性疫苗对肿瘤生长的抑制作用。根据公式计算肿瘤的体积。以GraphPad Prism制作肿瘤生长曲线图，如图8所示。统计学分析结果显示，Al(OH) 3对照组与PBS安慰剂组肿瘤生长曲线有一定差异。各MCV治疗性疫苗组肿瘤生长曲线与Vac疫苗组及Al(OH) 3对照组差异极为显著，值得注意的是，对照组R848/Al(OH) 3肿瘤生长受到抑制，CpG/Al(OH) 3组肿瘤生长曲线与Al(OH) 3对照组差异较显著，也有一定的抑制作用，与Vac疫苗组差异不显著。
这些结果说明本研究制备的MCV治疗性疫苗组具有不同程度的抗肿瘤作用，而佐剂在抗肿瘤作用中发挥着关键作用。
实施例12 MCV治疗性疫苗的抗肿瘤研究
本研究以R848，CpG等联合Al(OH) 3为佐剂制备了MCV治疗性疫苗，并开展MCV治疗性疫苗的抗肿瘤作用研究。
1.实验材料：CpG1826由上海捷瑞生物合成；R848购自invivogen公司(tlrl-r848)；氢氧化铝[Al(OH) 3]购自Brenntag。流式检测相关抗体购自Biolegend公司或ebioscience公司。
2.实验动物：实验中所使用的6-8周周龄的雌性BALB/c小鼠购自上海西普尔-必凯实验动物有限公司，饲养在上海西普尔-必凯实验动物有限公司SPF清洁级鼠房，所有动物实验 操作均在动物保护指导条例指导下进行。
3.实验过程：
3.1MCV肿瘤模型构建
在前期实验结果中，通过脂质体转染法将慢病毒质粒pcDH-VP1转染至BALB/c背景的肿瘤细胞CMS5中，在5μg/mL的Puromycin药物压力下筛选到了稳定表达VP1的细胞株CMS5-VP1/E2。将6-8周周龄的BALB/c雌性小鼠随机分笼，每组6只。通过向小鼠背部皮下接种1×10 6/100μL CMS5-VP1细胞悬液来构建MCV肿瘤模型。(见基础实施例1)
3.2MCV治疗性疫苗制备
将重组蛋白rVP1用缓冲液稀释后，以1:1的比例与铝佐剂Al(OH) 3混匀，4℃摇床中低速震荡过夜(以利于抗原均匀地吸附于Al(OH) 3佐剂)，作为MCV重组蛋白疫苗半成品。将MCV重组蛋白疫苗半成品与佐剂CpG、R848按一定比例混匀，4℃摇床中低速震荡2h，即为MCV治疗性疫苗。无论抗原和佐剂如何组合，使最终每单位疫苗(每只小鼠用量)rVP1含量为10μg，Al(OH) 3 500μg，CpG含量为10μg，R848含量为10μg。
3.3小鼠免疫
小鼠接种CMS5-VP1细胞系后第5，12，19天，分别制备各组疫苗进行肌肉免疫，免疫部位为小鼠后肢。在本实施例中小鼠共分为6组，包括：
PBS:0.1mL；
VP1/Al:rVP1 10μg；Al(OH) 3 500μg；0.1mL；
VP1/CpG/Al:rVP1 10μg；CpG 10μg；Al(OH) 3 500μg；0.1mL；
VP1/R848/Al:rVP1 10μg；R848 10μg；Al(OH) 3 500μg；0.1mL；
VP1/CpG/R848/Al:rVP1 10μg；CpG 10μg；R848 10μg；Al(OH) 3 500μg；0.1mL；
CpG/R848/Al:CpG 10μg；R848 10μg；Al(OH) 3 500μg；0.1mL；
3.4肿瘤生长曲线监测
CMS5细胞接种后第5天开始测量肿瘤的大小，每两天测量一次，并计算肿瘤的体积。以GraphPad Prism软件绘制肿瘤生长曲线图。肿瘤体积的计算公式为：Tumor volume(mm 3)＝L×W 2/2。
3.5细胞免疫检测
肿瘤接种后第33天将小鼠安乐死，摘取脾脏，制备单细胞悬液，以VP1抗原进行体外刺激后开展抗原特异性细胞免疫应答水平检测。并以GraphPad prism软件进行统计分析。
4.实验结果
4.1肿瘤接种后第33天各疫苗组小鼠荷瘤图片见图9。
4.2肿瘤接种后第33天各疫苗组荷瘤小鼠肿瘤组织见图10。
4.3肿瘤生长曲线见图11。
从实验结果来看，VP1/CpG/Al组及VP1/R848/Al组疫苗能够显著延缓CMS5-VP1荷瘤小鼠的生长，而VP1/CpG/R848/Al组与PBS未治疗组相比，生长曲线差异最为显著，通过该组疫苗治疗后，小鼠肿瘤基本得到控制，肿瘤大小基本没有生长且有被清除的趋势。该实验结果表明，CpG与R848佐剂无论是单一或者联合使用，都能提高VP1/Al疫苗对MCV肿瘤的治疗水平，其中，二者联合使用具有最为显著的抗肿瘤效果。
4.4疫苗诱导的抗原特异性细胞免疫应答
肿瘤接种后33天，通过流式细胞术检测小鼠脾脏中MCV疫苗诱导的抗原特异性细胞免疫应答，VP1/CpG/R848/Al疫苗组诱导CD4+T细胞分泌的IL-2、TNF-α、IFN-γ以及Granzyme B的水平、诱导CD8+T细胞分泌的IL-2、TNF-α、IFN-γ水平均明显高于其他疫苗组，即是说，VP1/CpG/R848/Al疫苗组通过激活机体的抗原特异性细胞免疫应答，T细胞通过分泌IL-2、TNF-α、IFN-γ、Granzyme B等效应因子来实现对肿瘤细胞的杀伤作用。
实施例13 MCV-VP1治疗性肿瘤疫苗佐剂筛选研究
1、MCV治疗性疫苗免疫学研究
预防性疫苗通常在发生病原体感染前接种，疫苗诱导宿主免疫***产生抗原特异性的抗体及免疫记忆，当病原体感染发生时，免疫***能有效地阻断病原体的入侵或者扩增。
如果宿主无法通过先天免疫***或者抗体介导的获得性免疫反应无法清除病原体，活化的T细胞通过其对已感染病原体的细胞的杀伤作用来抵御病原体的进一步感染。
在前期研究中，制备的MCV治疗性疫苗能明显地抑制肿瘤的生长。因此，通过动物实验来检测MCV治疗性疫苗是否通过增强T细胞免疫应答作用来实现对肿瘤的杀伤作用。免疫流程如下：分别于第0天和第14天注射，第28天取血。以含GM-CSF+IFN-α、TLR9激动剂CpG1826、TLR7/8激动剂R848及TLR4激动剂MPL为佐剂的MCV治疗性疫苗注射。
2、IV型超敏反应(DTH)
治疗性疫苗Vac/G+I、Vac/C、Vac/R、Vac/M及MCV重组蛋白疫苗Vac及Al(OH) 3佐剂对照组两次免疫程序(14天)，各组情况如下：
Vac/G+I组：10μg rVP1/10μg GM-CSF/1μg IFN-α/500μg Al(OH) 3；
Vac/C组：10μg rVP1/10μg CpG/500μg Al(OH) 3；
Vac/R组：10μg rVP1/10μg R848/500μg Al(OH) 3；
Vac/M组：10μg rVP1/25μg MPL/500μg Al(OH) 3；
Vac组：10μg rVP1/500μg Al(OH) 3；
Al(OH) 3组：500μg Al(OH) 3；
VP1组：10μg rVP1；
PBS组：10μLPBS。
免疫程序结束后第7天分别在小鼠的左、右后肢脚掌注射10μL的PBS或10μg rVP1(10μL)。
注射rVP1或PBS后24小时的脚掌肿胀如图12所示。从图中可以看出，接种MCV治疗性疫苗的小鼠脚掌明显肿胀。
根据公式计算各实验组脚掌肿胀厚度，以GraphPad Prism统计软件分析各疫苗组间的差异，DTH 24小时结果如图13所示。
从统计结果可以看出，Al(OH) 3或重组蛋白rVP1组的DTH反应不明显；MCV治疗性疫苗均表现较强的DTH反应，尤其是Vac/R疫苗组，与Vac差异极为显著。同时比较了Vac/G+I、Vac/C及Vac/M疫苗组与Vac/R疫苗的组间差异，Vac/C及Vac/M疫苗组与Vac/R疫苗组组间差异不显著，而Vac/R与Vac/G+I表现出极显著的组间差异。相比Vac组，各治疗性疫苗组中DTH反应最为强烈的为以R848为佐剂的Vac/R组，其次为以CpG为佐剂的Vac/C组以及以MPL为佐剂的Vac/M组，它们均表现出极显著的差异(****，p＜0.0001)，而以GM-CSF、IFN-α为佐剂的Vac/G+I的差异较为显著。这一结果与开展的MCV治疗性疫苗抑制肿瘤生长的结果较为一致。
DTH是由CD4 +或CD8 +T细胞介导的宿主对病原体或外源蛋白的免疫反应。因此，推测MCV治疗性疫苗可能通过诱导抗原特异性的T细胞免疫应答作用来实现抗肿瘤的功效。
为了确定MCV治疗性疫苗诱导的细胞免疫，在完成二次免疫后的第14天(及初次免疫后第28天)分别开展了血清学及细胞免疫学检测。
3、血清学检测
完成二次免疫后的第14天通过眼底采血并分离到小鼠血清。200ng/孔的rVP1包被96孔ELISA检测板，分离得到的小鼠血清为一抗，以制备的抗VP1兔多抗作为阳性对照，Al(OH) 3组血清作为阴性对照开展ELISA检测。
200ng/孔sVP1作为抗原包被96孔ELISA板。各样品血清以1:800为起始稀释度，连续对 倍稀释8个梯度，稀释的血清作为一抗、每孔100μL，每个稀释度两个复孔。以HRP标记的羊抗鼠作为二抗。
OD450/OD620nm读取光吸收值以阴性对照组的OD值的2.1倍作为cutoff值，实验组OD值>cut off即为阳性，相邻两个稀释度若一个为阳性，一个为阴性，则上一稀释度为该样品的抗体滴度。
血清学检测结果如图14所示，相比单独免疫抗原的VP1组，各疫苗组VP1特异性抗体滴度呈现出显著差异，而MCV治疗性疫苗组与MCV重组蛋白疫苗Vac间也有一定差异，尤其是Vac/C及Vac/M组，明显地高于Vac组。这说明Al(OH) 3能够增强宿主抗原特异性的体液反应。
4、细胞免疫学检测
肿瘤发生时由于肿瘤微环境的免疫抑制或免疫耐受作用，T细胞耗竭是临床治疗中的一大难题。因此设计治疗性疫苗时需要考虑的是能够增强宿主的细胞免疫。这也成为临床前研究中评价治疗性疫苗的重要指标。基于这些假设选择了GM-CSF+IFN-α、CpG、R848或MPL作为MCV治疗性疫苗的佐剂。因此在完成血清学检测后，开展了细胞免疫学检测已确定这些新型佐剂对细胞反应的刺激作用。
完成二次免疫后14天取小鼠脾细胞，1×10 6脾细胞以2μg重组蛋白体外刺激12小时后，以1×BFA封闭4小时。通过流式细胞术检测T细胞的TNF-α、IFN-γ、IL-2的表达水平，以验证MCV治疗性疫苗是否通过增强T细胞免疫反应来实现抗肿瘤的作用。框定淋巴细胞的单细胞，框定Fixable Viability Dye eFluor TM 780阴性的活细胞群后，框出CD3 +T细胞，分别检测CD4 +、CD8 +T细胞的TNF-α、IFN-γ及IL-2表达水平。
流式细胞术检测细胞因子方法：
对小鼠实施安乐死，取脾细胞，通过流式细胞术检测细胞因子。
①脾细胞制备
小鼠安乐死后，75％乙醇中浸泡10分钟。取脾脏，浸泡于RPMI1640完全培养基中。
以200目铜网研磨脾脏后，40μm孔径的细胞筛过滤。
1500rpm、4℃离心4分钟。弃上清，以1mL红细胞裂解液重悬细。
1500rpm、4℃离心4分钟。弃上清，以RPMI1640完全培养基洗涤2次。
1500rpm、4℃离心4分钟。弃上清，以RPMI1640完全培养基重悬细胞。
台盼蓝染色后，显微镜下细胞计数。
②脾细胞体外刺激
根据细胞计数结果，分别取1×10 6脾细胞悬液于96孔U底细胞培养板中。
加入2μg rVP1或加入1μg/mL Ion+0.1μg/mL PMA作为阳性对照，5％CO 2培养箱37℃培养12小时后，加入1×BFA封闭4小时。
封闭结束后，加入150μL 1×PBS终止体外刺激反应。4℃放置，备用。
③细胞因子染色及流式细胞术检测
取刺激过夜的脾细胞，1500rpm离心5分钟，甩去上清。
以含2％胎牛血清(FBS)的1×PBS重悬细胞，室温避光封闭15分钟。
表面染色：荧光标记的抗体稀释于含2％FBS的1×PBS中。封闭结束后1500rpm离心5分钟，甩去上清。以稀释好的抗体重悬细胞，室温避光染色15分钟。
固定：表面染色结束后，加入150μL 1×PBS以终止反应。1500rpm离心5分钟，甩去上清。加入200μL多聚甲醛，室温避光固定7分钟。
胞内染色：荧光标记的抗体稀释于含2％FBS的1×PBS中。多聚甲醛固定后，1500rpm离心5分钟，甩去上清。以1×PBS洗涤2次。以稀释好的抗体重悬细胞，室温避光染色15分钟。
流式细胞术检测：染色结束后，加入150μL 1×PBS以终止反应。1500rpm离心5分钟，甩去上清。以150μL 1×PBS重悬细胞。通过流式细胞术检测细胞因子表达水平。
④统计学分析
以统计学软件GraphPad Prism 6.0(GraphPad，La Jolla，CA，USA)对所有实验数据进行统计学分析，以平均值±标准平均值误差(SEM)表示。数据统计以t检验法比较各组之间的差异。nc(p＞0.05)表示无显著差异，*表示显著差异(p＜0.05)，**表示较显著差异(p＜0.01)，***表示非常显著差异(p＜0.001)，****表示极显著差异(p＜0.0001)。
流式细胞术检测结果：
(图15)表明，相比Vac、Al(OH) 3或VP1组，MCV治疗性疫苗组能诱导T细胞分泌TNF-α、IFN-γ及IL-2，Vac/R疫苗组TNF-α、IFN-γ及IL-2水平明显高于其他疫苗组。这与MCV治疗性疫苗抗肿瘤效果呈正相关。
这一研究结果说明，MCV治疗性疫苗诱导的T细胞免疫反应是通过CTL来实现对肿瘤细胞的杀伤作用。因此，认为MCV治疗性疫苗是通过诱导抗原特异性T细胞免疫反应来实现抗肿瘤效果的。
5、MCV治疗性疫苗机制探讨
在前期研究中发现在完成三次MCV治疗性疫苗接种后(肿瘤接种后第19-21天)肿瘤处于缓慢生长期。
6-8周周龄BALB/c小鼠接种CMS5-VP1肿瘤细胞后，肌肉注射MCV治疗性疫苗Vac/G+I、Vac/C、Vac/R、Vac/M，以及Vac，以及各对照组。完成3次疫苗接种后分别在肿瘤接种后第20、34天检测T细胞免疫反应。
CMS5-VP1/E2细胞以胰酶消化后制备成单细胞悬液，细胞计数，细胞活性率为95％。将细胞稀释至1×10 7/mL。
如图16所示，肿瘤接种后第19天Vac/R组肿瘤体积与其他疫苗组差异显著。每组各取3只小鼠，实施安乐死后，分别取小鼠脾脏制备脾细胞悬液，1×10 6的脾细胞以2μg rVP1刺激20小时，同时以1μg/mL离子霉素(Ionmycin)+0.1μg/mL佛波酯(PMA)刺激作为阳性对照。以流式细胞术检测抗原特异性细胞免疫反应，分别检测CD4 +、CD8 +T细胞分泌的TNF-α、IFN-γ及IL-2水平，如图17所示。Vac/R疫苗组CD4 +、CD8 +T细胞分泌的TNF-α、IFN-γ高于其他疫苗组，虽然CD4 +T细胞分泌的TNF-α及CD8 +T细胞分泌的IFN-γ没有统计学差异，这可能由于样本少而导致组间差异不明显。
肿瘤接种后第34天各疫苗组肿瘤体积组间差异显著，其中Vac/R疫苗对肿瘤的抑制作用尤为明显。各疫苗组小鼠实施安乐死后，取脾脏制备脾细胞悬液。1×10 6脾细胞以2μg rVP1刺激，同时以1μg/mL离子霉素(Ionmycin)+0.1μg/mL佛波酯(PMA)刺激作为阳性对照。流式细胞术检测CD4 +、CD8 +T细胞的TNF-α、IFN-γ及IL-2分泌水平。
如图18所示，相比Vac疫苗，MCV治疗性疫苗Vac/G+I、Vac/R、Vac/C及Vac/M可诱导抗原特异性细胞免疫反应，尤其是Vac/R疫苗诱导的抗原特异性细胞免疫反应最为显著，而Vac/R疫苗对肿瘤的抑制作用也最为显著。这些结果提示，MCV治疗性疫苗可能是通过诱导细胞免疫反应来实现对肿瘤细胞的杀伤作用。
6、小结
以GM-CSF+IFN-α、CpG1826、R848或MPL作为佐剂制备的MCV治疗性疫苗能有一定的抗肿瘤作用。同时，初步探讨了MCV治疗性疫苗的作用机制。研究发现，MCV治疗性疫苗能诱导抗原特异性的T细胞免疫反应。这些结果表明证实了本研究开发的MCV治疗性疫苗的可行性，同时也为MCV治疗性疫苗的临床研究提供了一定的数据支持。
实施例14 MCV治疗性疫苗免疫原性研究
前期研究采用了低温诱导表达的可溶性VP1(sVP1)制备的疫苗开展免疫原性研究。
为了获得大量的VP1，通过发酵罐高温诱导表达的VP1主要形成包涵体。为了确定重组装VP1(rVP1)是否具有与sVP1相当的免疫原性，将sVP1、rVP1吸附于Al(OH) 3佐剂，分别命名sVac、rVac。分别在第0、14天肌肉注射BALB/c小鼠，第28天眼眶取血，6000rpm离心10分钟分离血清，通过酶联免疫吸附法(ELISA)检测抗原特异性抗体滴度，具体方法如下：
包被：sVP1蛋白以抗原包被液稀释至2μg/mL，以稀释后的sVP1包被96孔ELISA板，100μL/孔，4℃孵育过夜。
封闭：抗原孵育过夜的96孔板甩掉抗原包被液，以1×PBST洗涤3次，加入含5％脱脂奶粉的1×PBST封闭液，150μL/孔，37℃封闭1小时。
一抗孵育：甩掉封闭液。以1×PBST洗涤5次。鼠血清以含2％脱脂奶粉的1×PBST稀释，起始稀释度1:200，连续对倍稀释8个梯度，以Al(OH) 3佐剂鼠血清作为阴性对照，100μL/孔，两个复孔。37℃孵育1小时。
二抗孵育：甩掉一抗孵育液。以1×PBST洗涤5次。HRP标记的羊抗鼠IgG以含2％脱脂奶粉的1×PBST稀释10000倍后，加入96孔板中，100μL/孔，37℃孵育1小时。
显色：甩掉二抗孵育液。以1×PBST洗涤5次。加TMB底物显色液，100μL/孔，37℃避光孵育5分钟。
终止：加入2M硫酸(H 2SO 4)终止显色反应，50μL/孔。
读数：OD 450/OD 620读取光吸收值。实验组OD值>cut off即为阳性，cutoff＝2.1×OD 对照组。
实验结果：
以sVP1或rVP1位抗原制备的疫苗sVP1-VAC或rVP1-VAC诱导的抗原特异性抗体滴度如图19所示。
从图19的抗体水平比较结果可看出，rVP1诱导的抗体滴度为sVP1的两倍。因此，采用重组装工艺制备的rVP1作为抗原开展MCV疫苗研究。
实施例15 MCV治疗性疫苗抗原特异性抗肿瘤作用
MCV治疗性疫苗应用在CMS5-VP1小鼠肿瘤模型上，能够达到使肿瘤完全消退的效果，为了确定该抗肿瘤效果是抗原特异性的，本研究通过向BALB/c小鼠接种乳腺癌细胞4T1来建立4T1小鼠肿瘤模型，并在该小鼠模型上使用MCV治疗性疫苗，以检测其是否具有抗原特异性。
实验材料：CpG1826由上海捷瑞生物合成；R848购自invivogen公司(tlrl-r848)；氢氧化铝[Al(OH) 3]购自Brenntag。
实验动物：实验中所使用的6-8周周龄的雌性BALB/c小鼠购自上海西普尔-必凯实验动物有限公司，饲养在上海西普尔-必凯实验动物有限公司SPF清洁级鼠房，所有动物实验操作均在动物保护指导条例指导下进行。
实验过程：
1、肿瘤模型建立
分别向BALB/c小鼠皮下左侧注射1×10 6/100μL CMS5-VP1，右侧注射1×10 6/100μL乳腺癌细胞4T1或纤维肉瘤细胞CMS5。
2、小鼠免疫
肿瘤接种后第五天将荷瘤小鼠随机分组，每组5只，分别于肿瘤接种后第5，12，19天左侧肌肉注射MCV治疗性疫苗(VP1/CpG/R848/Al(OH) 3)或不含VP1的疫苗(CpG/R848/Al(OH) 3)或PBS。
实验结果：
荷瘤小鼠CMS5-VP1肿瘤生长曲线如图20所示。荷瘤小鼠4T1肿瘤生长曲线如图21所示。
MCV治疗性疫苗对CMS5-VP1、CMS5、4T1三种肿瘤模型的抗肿瘤效果如图22所示。
从结果来看，CMS5-VP1/4T1荷瘤小鼠接种MCV治疗性疫苗后，左侧的CMS5-VP1疫苗完全消退，CpG/R848/铝佐剂组及PBS对照组的CMS5-VP1肿瘤明显，各疫苗组荷瘤小鼠右侧的4T1肿瘤明显，即是说，MCV治疗性疫苗不能完全抑制4T1肿瘤生长。结果同样表明，MCV治疗性疫苗仅能使CMS5-VP1肿瘤消退而对CMS5及4T1疫苗无治疗效果，即是说，MCV治疗性疫苗的抗肿瘤作用是特异性靶向表达VP1的CMS5-VP1肿瘤细胞的。
实施例16 2针或3针治疗性疫苗诱导的免疫持久性研究
在前期试验过程中，发现两针MCV治疗性疫苗已经能够起到使CMS5-VP1肿瘤完全消退的作用，因此在后续的实验中分别进行了2针及3针治疗性疫苗的免疫持久性探究。即在接种了CMS5-VP1的BALB/c肿瘤小鼠中免疫2针或3针的治疗性疫苗，末次免疫后以CMS5-VP1再挑战，并监测肿瘤生长。具体步骤如下：
1、实验材料：CpG1826由上海捷瑞生物合成；R848购自invivogen公司(tlrl-r848)；氢氧化铝[Al(OH) 3]购自Brenntag。
2、实验动物：实验中所使用的6-8周周龄的雌性BALB/c小鼠购自上海西普尔-必凯实验动物有限公司，饲养在上海西普尔-必凯实验动物有限公司SPF清洁级鼠房，所有动物实验操作均在动物保护指导条例指导下进行。
3、肿瘤模型建立：分别向BALB/c小鼠皮下注射1×10 6/100μL CMS5-VP1。
4、2针免疫：肿瘤接种后第五天将荷瘤小鼠随机分组，每组8只，分别于肿瘤接种后第5，12天肌肉注射疫苗。小鼠共分为5组，包括：
PBS：0.1mL；
VP1/CpG/Al(OH) 3：rVP1 10μg；CpG 10μg；Al(OH) 3 500μg；0.1mL；
VP1/R848/Al(OH) 3：rVP1 10μg；R848 10μg；Al(OH) 3 500μg；0.1mL；
VP1/CpG/R848/Al(OH) 3：rVP1 10μg；CpG 10μg；R848 10μg；Al(OH) 3 500μg；0.1mL；
VP1/Al(OH) 3：rVP1 10μg；Al(OH) 3 500μg；0.1mL；
3针免疫：肿瘤接种后第五天将荷瘤小鼠随机分组，每组8只，分别于肿瘤接种后第5，12，19天肌肉注射疫苗。小鼠共分为4组，包括：
PBS：0.1mL；
CpG/R848/Al(OH) 3：CpG 10μg；R848 10μg；Al(OH) 3 500μg；0.1mL；
VP1/CpG/R848/Al(OH) 3：rVP1 10μg；CpG 10μg；R848 10μg；Al(OH) 3 500μg；0.1mL；
VP1/Al(OH) 3：rVP1 10μg；Al(OH) 3 500μg；0.1mL；
5、CM5-VP1肿瘤细胞再挑战：末次免疫后30天(2针：第42天；3针：第49天)对MCV治疗疫苗免疫组小鼠背部再次皮下注射1×10 6/100μL CMS5-VP1细胞。
6、实验结果：各疫苗组2针及3针接种疫苗后CMS5-VP1荷瘤小鼠肿瘤生长曲线(左两针，右三针)。
结果表明，不管是两针还是三针的MCV治疗性疫苗，均能使MCV-VP1荷瘤小鼠肿瘤完全消退。
末次免疫后CMS5-VP1肿瘤再挑战实验肿瘤生长曲线(左两针右三针)。
结果表明，虽然两针及三针的治疗性疫苗均能够室肿瘤完全消退，但三针疫苗的免疫持久性更好。
实施例17 MCV治疗性疫苗抗肿瘤作用机制研究
前期研究表明MCV治疗性疫苗能够诱导机体抗原特异性细胞免疫应答反应来实现抗肿瘤效果，为了进一步确定相关机制，本研究以anti-CD3，anti-CD4或anti-CD8抗体封闭T细胞，同时检测疫苗免疫后小鼠肿瘤生长曲线。
1、实验材料：CpG1826由上海捷瑞生物合成；R848购自invivogen公司(tlrl-r848)； 氢氧化铝[Al(OH) 3]购自Brenntag。小鼠抗体anti-CD3、anti-CD4、anti-CD8购自BioxCell。
2、实验动物：实验中所使用的6-8周周龄的雌性BALB/c小鼠购自上海西普尔-必凯实验动物有限公司，饲养在上海西普尔-必凯实验动物有限公司SPF清洁级鼠房，所有动物实验操作均在动物保护指导条例指导下进行。
3、肿瘤模型建立：分别向BALB/c小鼠皮下注射1×10 6/100μL CMS5-VP1。
4、小鼠免疫：肿瘤接种后第4天将荷瘤小鼠随机分组，共5组，每组5只，其中三组荷瘤小鼠分别于肿瘤接种后第4，11，18天腹腔注射200μg anti-CD3、anti-CD4、anti-CD8单抗以封闭CD3，CD4，CD8T细胞，注射抗体后24小时(肿瘤接种后第5，12，19天)，肌肉注射MCV治疗性疫苗(VP1/CpG/R848/Al(OH) 3)或PBS。
4、实验结果：肿瘤生长曲线如图23所示。
从结果来看，用CD3，CD4，CD8单抗封闭相应T细胞后减弱MCV治疗性疫苗的抗肿瘤效果，即是说，T细胞对MCV治疗性疫苗的抗肿瘤效果的实现是很关键的。
实施例18 MCV治疗性疫苗抗肿瘤效果与Treg的相关性
1、实验材料：CpG1826由上海捷瑞生物合成；R848购自invivogen公司(tlrl-r848)；氢氧化铝[Al(OH) 3]购自Brenntag。
2、实验动物：实验中所使用的6-8周周龄的雌性BALB/c小鼠购自上海西普尔-必凯实验动物有限公司，饲养在上海西普尔-必凯实验动物有限公司SPF清洁级鼠房，所有动物实验操作均在动物保护指导条例指导下进行。
3、肿瘤模型建立参照实验例1。
4、小鼠免疫：肿瘤接种后第五天，将小鼠随机分为三组，每组各五只，分别于第5，12，19天肌肉免疫小鼠，每组按照下述组分：
PBS：0.1mL；
VP1/CpG/R848/Al(OH) 3：rVP1 10μg；CpG 10μg；R848 10μg；Al(OH) 3 500μg；0.1mL；
VP1/Al(OH) 3：rVP1 10μg；Al(OH) 3 500μg；0.1mL；
5、流式检测：肿瘤接种后第33天，牺牲小鼠，取出脾脏，制备单细胞悬液，并利用流式细胞术检测各疫苗免疫组Treg相关细胞因子表达。
6、实验结果
各疫苗组荷瘤小鼠***中Treg的TGF-β表达水平如图24所示。
从实验结果来看，MCV治疗性疫苗(VP1/CpG/R848/Al(OH) 3)免疫组小鼠Treg细胞表达TGF-β水平显著低于其他实验组，预示着MCV治疗性疫苗免疫后，能够抑制Treg细胞反应，增强T细胞免疫反应，从而达到抗肿瘤效果。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (15)

  1. 一种针对默克尔细胞癌的疫苗组合物，其特征在于，所述疫苗组合物包含默克尔细胞癌多瘤病毒衣壳蛋白VP1或包含编码默克尔细胞癌多瘤病毒衣壳蛋白VP1的基因的质粒；以及TLR激动剂，所述TLR激动剂选自CpG、R848和MPL中的一种或多种。
  2. 根据权利要求1所述的疫苗组合物，其特征在于，所述TLR激动剂为CpG和R848的组合。
  3. 根据权利要求1或者2所述的疫苗组合物，其特征在于，所述组合物进一步包含铝佐剂。
  4. 根据权利要求3所述的疫苗组合物，其特征在于，所述疫苗包括5-500重量份的默克尔细胞癌多瘤病毒衣壳蛋白VP1或包含编码默克尔细胞癌多瘤病毒衣壳蛋白VP1的基因的质粒、2.5-250重量份的TLR激动剂和10-1000重量份的铝佐剂。
  5. 根据权利要求4所述的疫苗组合物，其特征在于，所述疫苗包括10-100默克尔细胞癌多瘤病毒衣壳蛋白VP1或包含编码默克尔细胞癌多瘤病毒衣壳蛋白VP1的基因的质粒、5-100重量份的TLR激动剂和50-500重量份的铝佐剂。
  6. 根据权利要求1或者2所述的疫苗组合物，其特征在于：所述的VP1的核苷酸序列与SEQ ID No.6具有71.9-74.7％同源性。
  7. 根据权利要求1或者2所述的疫苗组合物，其特征在于：所述的VP1的核苷酸序列为SEQ ID No.1、SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示。
  8. 一种默克尔细胞癌多瘤病毒衣壳蛋白VP1的编码基因，其特征在于：所述的编码基因与SEQ ID No.6具有71.9-74.7％同源性的核苷酸序列。
  9. 根据权利要求8所述的编码基因，其特征在于：所述的编码基因的序列为SEQ ID No.1、SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示的核苷酸序列。
  10. 权利要求8或9所述的编码基因在制备针对默克尔细胞癌的疫苗中的应用。
  11. 根据权利要求10所述的应用，其特征在于：利用SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示的核苷酸序列制备DNA疫苗或蛋白质疫苗。
  12. 一种针对默克尔细胞癌的疫苗，其特征在于：所述疫苗利用权利要求8所述的编码基因制得。
  13. 根据权利要求12所述的疫苗，其特征在于：所述的疫苗为DNA疫苗或蛋白疫苗，所述的DNA疫苗包含SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示的核苷酸序列或其突变型中的一种或多种；所述的蛋白质疫苗包含SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4所示的核苷酸序列编码的蛋白质或其突变体中的一种或多种。
  14. 根据权利要求13所述的疫苗，其特征在于：所述疫苗还包含佐剂，所述佐剂包括TLR激动剂和铝佐剂，所述TLR激动剂选自CpG、R848和MPL中的一种或多种。
  15. 根据权利要求12-14任一项所述的疫苗，其特征在于：所述的疫苗为预防性和/或治疗性疫苗。
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