重组表位疫苗的制备及免疫学活性研究
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摘要
重组表位疫苗(Recombinant epitope vaccine)研制和开发过程主要包括构建表达、制备及活性研究等几个阶段。围绕以上三个方面,针对重组表位疫苗CZ-1、HSP65-MUC1和HSP65-HER2进行了以下研究:1、使用分子生物学软件对AsiaⅠ型口蹄疫病毒的VP1上的B细胞表位和Th细胞表位进行了多参数预测,根据预测结果在大肠杆菌内构建表达了由相同B细胞表位及Th细胞表位以串联模式组成口蹄疫重组表位疫苗CZ-1,采用层析法纯化、复性后,对其免疫豚鼠后产生中和抗体的滴度进行了测定;2、解决了HSP65-MUC1的降解问题,建立了重组表位疫苗HSP65-MUC1的实验室规模纯化工艺,并将纯化工艺放大至中试规模后,且对纯化后的HSP65-MUC1原液冻干品进行了一系列的质量检测;3、表达并纯化了重组表位疫苗HSP65-HER2,采用小鼠B16/HER2黑色素瘤的腹腔荷瘤动物模型研究其生物学活性,并对其作用机制进行了初步探索。
本研究的意义在于,构建、表达、纯化获得了重组表位疫苗CZ-1,并对其生物学活性进行了研究,证实其有望开发成预防AsiaⅠ型FDMV感染的新型疫苗,并为其他同类型的重组表位疫苗的开发提供了思路;解决了HSP65-MUC1在制备过程中易降解的技术难题,建立了HSP65-MUC1的中试工艺,为进一步临床试验及商业化应用建立了坚实的基础;通过小鼠体内抑瘤实验,证实了HSP65-HER2与CpG ODN联合应用对HER2阳性肿瘤细胞生长抑制作用,为进一步的研究奠定了基础。
Recombinant epitope vaccine is a kind of vaccine which is comprised of recombinant epitopes and prepared by genetic engineering technology. The epitope- vaccine is consist of antigen specific epitopes which can induce specific immunogenic response against pathogens. It overcomes the crucial drawbacks of traditional vaccines which based on the intact pathogen and could be approach of safer vaccines.
Specificity, construction, preparation and activity are crucial for the development of such epitope-based vaccines. In this study, we focus on the following concepts: first, identification of the B-cell epitopes in VP1 subunit of Foot-and-mouth disease virus (FMDV), preparation of the AsiaⅠtype FMDV epitope-vaccine CZ-1 and determination of the activity; Second, establishment of the lab-scale purification process of a HSP65-MUC1 protein, scaling it up to pilot-scale and quality control of the purified HSP65-MUC1 according to the Chinese Pharmacopoeia 2005; third, establishment of HER2 positive B16 cell line, preparation of HSP65-HER2 recombinant fusion protein and determination of its immunogenic activity on mice mode.
This study was composed of following parts:
1.1 Construction and preparation of AsiaⅠtype FMDV epitope-vaccine
CZ-1 and determination of its immunogenicity.
The VP1 subunits of FMDV is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. B cell epitopes and T helper cell eptiopes in VP1 subunit was predicted by multi-parameters method using DNAStar software. The cDNA sequence encoding the B cell epitope and T helper cell epitope peptide was constructed artificially by PCR and then three units was connected in series pattern. The cDNA was cloned into the vector pET28a to construct pET28-CZ-1 plasmid, which was transformed into E.coli B21(DE3). After induced by IPTG, the CZ-1 was expressed as inclusion body which was solubilized in buffer of 8mol/L urea, and was purified by Ni-affinity chromatography and gel chromatography. In the purification process, the CZ-1 was refolded by decreasing the concentration of urea in the washing buffers in a stepwise gradient manner. 74 mg of CZ-1 with purity of 90% was purified from 12.5 g wet cell mass. Guinea pigs were immunized with CZ-1 and sera were collected. Neutralizing antibodies in the sera were determined by neutralization test in cells and protection assay in neonate rats. The antibody titer elicited by CZ-1 was 1:286 and 1:127 in cell neutralization test and neonate rats protection assay respectively, which was comparable to the traditional vaccines of whole pathogen.
1.2 Establishment of the HSP65-MUC1 pilot-scale purification scheme and detection of the purified HSP65-MUC1
HSP65-MUC1, a fusion protein between Bacillus Calmette-Guerin (BCG) derived heat shock protein 65 (HSP65) and human MUC1 VNTR peptides, was approved by China’s FDA to initiate a phase clinical trial for the treatment of patients with MUC1-positive breast cancer. To avoid the degradation of HSP65-MUC1, we increased the pH and the NaCl concentration of the buffers used in homogenization and HIC to 9.0 that was demonstrated crucial for stabilizing HSP65-MUC1. To produce qualified HSP65-MUC1, a lab-scale purification scheme of HSP65-MUC1 was established. The scheme consists of two steps including hydrophobic interaction chromatography (HIC) and ion-exchange chromatography (IEX). The purity of purified HSP65-MUC1 was up to 95%. And then, the lab-scale purification scheme was scaled up to pilot-scale by adjusting the NaCl concentration of elution buffer in HIC. In pilot scale, 400mg HSP65-MUC1 with purity of 96% was purified from 100 g wet cell mass. According to the Chinese pharmacopeia, the quality control method of HSP65-MUC1 was set up. To detect the general properties (appearance and pH), purity and sterility, we reconstituted lyophilized HSP65-MUC1 in a vial with 0.5ml Mili-Q water. The reconstituted sample, without color or particles, with pH 7.1, was clear. The concentration of HSP65-MUC1 was 0.996mg/ml. The purity of HSP65-MUC1 was 96%. The molecular weight and pI of HSP65-MUC1 were 64 kDa and 4.8, respectively. Endotoxin level of HSP65-MUC1 was less than 0.25 EU/mg, and no rabbit showed a temperature rise of 0.5°C compared with respective control temperature at any time period. E.coil protein and DNA were less than 10 ng/mg protein and less than 100 pg/mg protein, respectively. No microbial growth was observed for 14 days. The amino sequence obtained by N-teminal sequencing matched exactly the predicted N-termini of the HSP65-MUC1. The HSP65-MUC1 stored over 12 months at 4°C showed no evident degradation, aggregation, and potency loss.
In a word, the purified HSP65-MUC1 meet the requirement of phaseⅠc linical trials.
1.3 Study on immunologic activity of HSP65-HER2
HSP65-HER2 is a fusion protein between Bacillus Calmette-Guerin (BCG) derived heat shock protein 65 (HSP65) and multi- epitopes. In the previous reports, HSP65-HER2 can induce the specific and non-specific immune response to inhibit the generation of ZR-75-30 (HER2 positive). The immunologic activity of HSP65-HER2 was verified further by the tumor growth inhibition in mice model.
After screening culture by G418 and monoclonal culture, the stable expressed HER2 B16 cell line was established with the transfection of the pCDNA3-GFP-HER2 plasmid, and the expression of HER2 gene were identified by fluorescence microscope and flow cytometry. HSP65-HER2 was expressed in E.coli, purified by Ni affinity chromatography and superdex G-25, and the endotoxin was removed by Triton X-114.
In the study of the prophylactic effect of HSP65-HER2, inhibition of the growth of HER2-expressing tumors and prolonged survival of tumor-bearing mice were observed in the HSP65-HER2 groups immunized with or without CpG ODN as adjuvant. And in the therapeutic study, inhibition of the growth of HER2-expressing tumors and prolongation of the survival of tumor-bearing mice were observed in the groups immunized with HSP65-HER2 plus CpG ODN as adjuvant, while the no such effect was observed in the groups immunized with only HSP65-HER2 or only CpG ODN. HSP65-HER2 inhibit the growth of HER2- expressing tumors by inducing the HER2-specific CTL.
In the study, we constructed and prepared the recombinant epitope vaccine CZ-1, identified the immunogenicity by neutralization test in cell line and protection assay in neonate rats, established the pilot-scale purification scheme, prepared sufficient clinical grade HSP65-MUC1, and identified the tumor growth inhibition effect of HSP65-HER2 in mice model.
本研究的意义在于,构建、表达、纯化获得了重组表位疫苗CZ-1,并对其生物学活性进行了研究,证实其有望开发成预防AsiaⅠ型FDMV感染的新型疫苗,并为其他同类型的重组表位疫苗的开发提供了思路;解决了HSP65-MUC1在制备过程中易降解的技术难题,建立了HSP65-MUC1的中试工艺,为进一步临床试验及商业化应用建立了坚实的基础;通过小鼠体内抑瘤实验,证实了HSP65-HER2与CpG ODN联合应用对HER2阳性肿瘤细胞生长抑制作用,为进一步的研究奠定了基础。
Recombinant epitope vaccine is a kind of vaccine which is comprised of recombinant epitopes and prepared by genetic engineering technology. The epitope- vaccine is consist of antigen specific epitopes which can induce specific immunogenic response against pathogens. It overcomes the crucial drawbacks of traditional vaccines which based on the intact pathogen and could be approach of safer vaccines.
Specificity, construction, preparation and activity are crucial for the development of such epitope-based vaccines. In this study, we focus on the following concepts: first, identification of the B-cell epitopes in VP1 subunit of Foot-and-mouth disease virus (FMDV), preparation of the AsiaⅠtype FMDV epitope-vaccine CZ-1 and determination of the activity; Second, establishment of the lab-scale purification process of a HSP65-MUC1 protein, scaling it up to pilot-scale and quality control of the purified HSP65-MUC1 according to the Chinese Pharmacopoeia 2005; third, establishment of HER2 positive B16 cell line, preparation of HSP65-HER2 recombinant fusion protein and determination of its immunogenic activity on mice mode.
This study was composed of following parts:
1.1 Construction and preparation of AsiaⅠtype FMDV epitope-vaccine
CZ-1 and determination of its immunogenicity.
The VP1 subunits of FMDV is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. B cell epitopes and T helper cell eptiopes in VP1 subunit was predicted by multi-parameters method using DNAStar software. The cDNA sequence encoding the B cell epitope and T helper cell epitope peptide was constructed artificially by PCR and then three units was connected in series pattern. The cDNA was cloned into the vector pET28a to construct pET28-CZ-1 plasmid, which was transformed into E.coli B21(DE3). After induced by IPTG, the CZ-1 was expressed as inclusion body which was solubilized in buffer of 8mol/L urea, and was purified by Ni-affinity chromatography and gel chromatography. In the purification process, the CZ-1 was refolded by decreasing the concentration of urea in the washing buffers in a stepwise gradient manner. 74 mg of CZ-1 with purity of 90% was purified from 12.5 g wet cell mass. Guinea pigs were immunized with CZ-1 and sera were collected. Neutralizing antibodies in the sera were determined by neutralization test in cells and protection assay in neonate rats. The antibody titer elicited by CZ-1 was 1:286 and 1:127 in cell neutralization test and neonate rats protection assay respectively, which was comparable to the traditional vaccines of whole pathogen.
1.2 Establishment of the HSP65-MUC1 pilot-scale purification scheme and detection of the purified HSP65-MUC1
HSP65-MUC1, a fusion protein between Bacillus Calmette-Guerin (BCG) derived heat shock protein 65 (HSP65) and human MUC1 VNTR peptides, was approved by China’s FDA to initiate a phase clinical trial for the treatment of patients with MUC1-positive breast cancer. To avoid the degradation of HSP65-MUC1, we increased the pH and the NaCl concentration of the buffers used in homogenization and HIC to 9.0 that was demonstrated crucial for stabilizing HSP65-MUC1. To produce qualified HSP65-MUC1, a lab-scale purification scheme of HSP65-MUC1 was established. The scheme consists of two steps including hydrophobic interaction chromatography (HIC) and ion-exchange chromatography (IEX). The purity of purified HSP65-MUC1 was up to 95%. And then, the lab-scale purification scheme was scaled up to pilot-scale by adjusting the NaCl concentration of elution buffer in HIC. In pilot scale, 400mg HSP65-MUC1 with purity of 96% was purified from 100 g wet cell mass. According to the Chinese pharmacopeia, the quality control method of HSP65-MUC1 was set up. To detect the general properties (appearance and pH), purity and sterility, we reconstituted lyophilized HSP65-MUC1 in a vial with 0.5ml Mili-Q water. The reconstituted sample, without color or particles, with pH 7.1, was clear. The concentration of HSP65-MUC1 was 0.996mg/ml. The purity of HSP65-MUC1 was 96%. The molecular weight and pI of HSP65-MUC1 were 64 kDa and 4.8, respectively. Endotoxin level of HSP65-MUC1 was less than 0.25 EU/mg, and no rabbit showed a temperature rise of 0.5°C compared with respective control temperature at any time period. E.coil protein and DNA were less than 10 ng/mg protein and less than 100 pg/mg protein, respectively. No microbial growth was observed for 14 days. The amino sequence obtained by N-teminal sequencing matched exactly the predicted N-termini of the HSP65-MUC1. The HSP65-MUC1 stored over 12 months at 4°C showed no evident degradation, aggregation, and potency loss.
In a word, the purified HSP65-MUC1 meet the requirement of phaseⅠc linical trials.
1.3 Study on immunologic activity of HSP65-HER2
HSP65-HER2 is a fusion protein between Bacillus Calmette-Guerin (BCG) derived heat shock protein 65 (HSP65) and multi- epitopes. In the previous reports, HSP65-HER2 can induce the specific and non-specific immune response to inhibit the generation of ZR-75-30 (HER2 positive). The immunologic activity of HSP65-HER2 was verified further by the tumor growth inhibition in mice model.
After screening culture by G418 and monoclonal culture, the stable expressed HER2 B16 cell line was established with the transfection of the pCDNA3-GFP-HER2 plasmid, and the expression of HER2 gene were identified by fluorescence microscope and flow cytometry. HSP65-HER2 was expressed in E.coli, purified by Ni affinity chromatography and superdex G-25, and the endotoxin was removed by Triton X-114.
In the study of the prophylactic effect of HSP65-HER2, inhibition of the growth of HER2-expressing tumors and prolonged survival of tumor-bearing mice were observed in the HSP65-HER2 groups immunized with or without CpG ODN as adjuvant. And in the therapeutic study, inhibition of the growth of HER2-expressing tumors and prolongation of the survival of tumor-bearing mice were observed in the groups immunized with HSP65-HER2 plus CpG ODN as adjuvant, while the no such effect was observed in the groups immunized with only HSP65-HER2 or only CpG ODN. HSP65-HER2 inhibit the growth of HER2- expressing tumors by inducing the HER2-specific CTL.
In the study, we constructed and prepared the recombinant epitope vaccine CZ-1, identified the immunogenicity by neutralization test in cell line and protection assay in neonate rats, established the pilot-scale purification scheme, prepared sufficient clinical grade HSP65-MUC1, and identified the tumor growth inhibition effect of HSP65-HER2 in mice model.
引文
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