HIV复合表位核酸和痘苗病毒载体疫苗构建及免疫原性研究
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摘要
鉴于当前获得性免疫缺陷综合征（Acquired immunodeficiency syndrome, AIDS）的流行态势和预防治疗现状，研发安全有效的艾滋病预防/治疗性疫苗刻不容缓。近些年来，很多科学家利用不同的疫苗载体和形式、不同的人免疫缺陷病毒（Human immunodeficiency virus，HIV）免疫原、不同的接种或免疫途径进行了大量的有益的尝试。本研究选择DNA质粒和痘苗病毒天坛株作为免疫原递送系统，开展艾滋病疫苗的免疫原性研究。
本研究在本研究室多年来筛选获得的HIV复合多表位基因（MEGNp24）的基础上，在其上游引入CpG基序和CTB基因作为佐剂，构建了一种重组核酸疫苗pVL-CCMp24，将重组DNA质粒转染293T细胞，从RT-PCR和间接免疫荧光检测结果可知，重组质粒编码的目的基因CCMp24在哺乳动物细胞内得到正确转录和表达。肌肉注射途径免疫BALB/c小鼠，分析重组DNA疫苗的免疫原性，由对HIV特异性抗体、Th1型和Th2型细胞因子、T细胞亚型、淋巴细胞增殖等免疫指标的检测结果表明，重组DNA候选疫苗pVL-CCMp24可以诱导机体产生一定程度的细胞和体液免疫应答。
基于多价疫苗的设计理念，本研究首先构建了一种含三个表达盒的痘苗病毒穿梭载体pSTKE，含有三个独立的外源基因表达盒，分别以VTT及其基因缺失突变株作为原始病毒，以增强型绿色荧光蛋白(EGFP)、红色荧光蛋白（RFP）和蓝色荧光蛋白（BFP）作为报告基因，分别对三个表达盒的功能进行验证。通过噬斑筛选，获得两株重组痘苗病毒(rVTT-EGFP、rdVTT-EGFP)，利用PCR、实时荧光定量PCR、Western blot方法对重组病毒以及对外源基因的表达量和遗传稳定性进行分析。结果表明，成功构建了三基因表达盒痘苗病毒穿梭载体，成功筛选出两株重组痘苗病毒，EGFP基因在痘苗病毒内得到高水平表达，三个表达盒之间没有相互影响，而且重组病毒具有良好的遗传稳定性。
将重组核酸疫苗pVL-CCMp24上的目的免疫原基因CCMp24连接到痘苗病毒穿梭载体pSTKE的第一个表达盒内即MCS1，由特异性引物扩增获得的含Loxp基因序列的EGFP基因（EGFP基因两端的Loxp基因方向相同）克隆到pSTKE的第三个表达盒MCS3中，重组质粒pCCMp24-LEL鉴定正确后，转染dVTT感染的BHK-21细胞，通过同源重组、病毒蚀斑筛选获得重组CCMp24和EGFP的E3L及TK基因缺失的痘苗病毒rddVTT-CCMp24G，然后利用Cre/Loxp重组系统将EGFP基因敲除，获得仅表达目的免疫原基因的双基因缺失重组痘苗病毒rddVTT-CCMp24。RT-PCR、间接免疫荧光、Western Blot检测结果表明，CCMp24融合基因在痘苗病毒感染的BHK-21细胞中得到高效转录和表达。
痘苗病毒在长期的实践应用中具有毒副作用，存在安全隐患。本研究在缺失病毒毒力相关基因的痘苗病毒的基础上，通过痘苗病毒穿梭载体pSTKE携带的目的免疫原基因将TK基因缺失，筛选的基因缺失型重组痘苗病毒通过鼻腔感染和颅内注射感染途径感染BALB/c小鼠，通过监测感染小鼠的体征变化、体重变化、病毒感染后的死亡率等方面评价E3L基因和TK基因缺失的重组痘苗病毒在小鼠体内的毒性。监测结果表明，重组病毒rddVTT-CCMp24的毒力较野生型VTT有很大程度的减弱，提高了进一步研究或应用的安全性。说明缺失E3L基因和TK基因可以使痘苗病毒致弱，且外源基因的引入对病毒的毒力没有显著影响。
在了解其毒力的基础上，以BALB/c小鼠为模型开展了重组病毒rddVTT-CCMp24的免疫原性分析。按照既定的免疫程序免疫后，通过对抗体、细胞因子及T细胞亚型等各项指标的检测，结果显示，rddVTT-CCMp24可诱导机体产生分泌IFN-γ的T细胞数量显著增加，CD4+和CD8+T细胞数增加，脾细胞体外接受HIV特异性抗原刺激下增殖能力显著提高。HIV特异性抗体检测结果显示，可诱导较高的HIV特异性抗体水平，且诱导IL-2和IL-4细胞因子的分泌。综合结果可知，重组病毒可激发机体细胞和体液免疫应答。
同时，采用DNA/rddVTT-CCMp24prime-boost免疫策略在小鼠模型上检测免疫效果，结果相对于DNA疫苗或rddVTT-CCMp24疫苗分别单独免疫，可更好的刺激分泌IFN-γ产生T细胞的分化和增殖，IFN-γ ELISPOT检测结果显示，HIV特异性的分泌IFN-γ的记忆性T细胞数量显著高于单独免疫组。表明，prime-boost免疫策略诱导较强的免疫应答和免疫记忆形成，为下一步的实验研究提供了数据支持。
Respecting the currently epidemic of AIDS and its status quo of prevention and therapy, it isimminent to develop a safe and effective AIDS preventative or therapic vaccine. In these years,many scientists had carried out a large number of beneficial trials with various vaccine vectors andmodalities, HIV immunogens, or different routes of vaccination and immunifaction. In thisinvestigation, DNA plasmid and vaccinia virus Tian Tan strain as immunogen delivery systemwere used to launch the trial research of vaccines against HIV.
In this study, we firstly constructed a eukaryotic expression vector of HIV multi-epitope genecontaining unmethylated CpG ODN sequence and cholera toxin B subunit (CTB) and expressionin vitro. CpG-CTB gene sequence (CC) was obtained by PCR using the primers of CTB genecontaining CpG ODN and Linker Sequence designed, and then was connected with pVL. HIVmulti-epitope gene (MEGNp24) from pVAX1-MEGNp24was inserted into the downstream of CCgene to construct pVL-CCMp24. After the recombination plasmid pVL-CCMp24was confirmedby enzyme digestion, it transfected into293T cells with liposome. The expression of CCMp24protein was confirmed by RT-PCR and indirect immunofluorescence. The results showed that theCTB and HIV multi-epitope gene were stably expressed in the293T cells transfected. And thenBALB/c mice were injected with the DNA vaccine by intramuscular injection, to evaluate theimmunogenicity of recombinant DNA vaccine. The immune effects induced by pVL-CCMp24were detected by measuring HIV-specific antibody, Th1type and Th2type cytokines in bloodserum, T cells subtype and lymphocyte proliferation. The results suggested that the recombinantDNA vaccine pVL-CCMp24can induced the production of cellular and humoral immunityresponses in some degree.
Based on the idea of multivalent vaccines and preliminary findings, this study designed andconstructed a vaccinia virus shuttle vector pSTKE with Triple-gene expression cassette. Thevector used vaccinia virus thymidine kinase gene (TK) as screening marker, and contained threeseparate foreign gene expression cassettes. The derived recombinant vaccinia virus can express atleast three different target genes with high efficiency at the whole early and late stages. In addition,knocking out of TK attenuated the virus and improved the safety of clinical application. UsingChinese vaccinia virus Tian Tan strain (VTT) and vaccinia virus Tian Tan strain deleting E3Lgene (dVTT) as original viruses, and enhanced green fluorescent protein (EGFP), red fluorescentprotein (RFP) and blue fluorescent protein (BFP) as reporter gene, we verified the three expressioncassettes. By plaque screening, two recombinant vaccinia viruses (rVTT-EGFP and rdVTT-EGFP)were obtained, and the expression and genetic stability of recombinant virus and foreign genes were analyzed using PCR, real-time quantitative PCR, and Western blot. Results showedsuccessful construction of vaccinia virus shuttle vector with three-gene expression cassette. Twostrains of two recombinant vaccinia viruses were identified, EGFP gene was expressed in thevaccinia virus with a high level, and there was no interaction between the three expressioncassettes, and the two recombinant viruses had good genetic stability.
A HIV immunogen (CCMp24) from pVL-CCMp24was first cloned into MCS1of vacciniavirus shuttle vector pSTKE, and a fragment (LoxP-EGFP-LoxP) containing Loxp sequence andEGFP gene obtained by PCR with the specific primers was inserted into MCS3of shuttle vectorpSTKE, resulting in the recombinant plasmid pCCMp24-LEL. the recombinant plasmid wastransfected into BHK21cells infected with dVTT, and the recombinant virus rddVTT-CCMp24Gwaspurified with plaque isolates expressing green fluorescent protein used as a selection marker, andEGFP gene in the genome of rddVTT-CCMp24Gwas deleted based on Cre/loxp site-specificrecombination, and plaque screeing was performed as described above to generate therecombinant vaccinia virus rddVTT-CCMp24. RT-PCR, indirect immunofluorescence and Westernblot were used to detected the expression of CCMp24in BHK-21cells infected by rddVTT-CCMp24,the results demonsteated that CTB and HIV MEGNp24were expressed in BHK-21cells.
In this research, based on vaccinia virus of virulence related gene deletion, the deletion of TKwas implemented by homologous recombination. The virulence of recombinant virusrddVTT-CCMp24was evaluated in inbred BALB/c mice by the daily measurement of body weightchange after the mice were infected intranasally with rddVTT-CCMp24, ddVTT or VTT.Simultaneously, the neurovirulence of rddVTT-CCMp24was assessed by challenging BALB/c micevia intraperitoneal injection. The neurovirulence in infected mice was determined by measuring50%of the intracranial lethal infectious dose (ICLD50). These results suggest that rddVTT-CCMp24and ddVTT were attenuated due to the deletion of E3L and TK genes, and the introduction of CTBand HIV MEGNp24genes doesn’t alter the infectivity and virulence of the vaccinia virus vectors.
Subsequently, in BALB/c models, the immunogenicity of rddVTT-CCMp24was analyzedthrough measuring HIV-specific antiboady, cytokines in peripheral blood, cellular surface antigenof splenocytes and the number of T cells secreting IFN-γ. We found that the expression of thisimmunogen was sufficient to facilitate the development of substantial number of antigen-specificT cells secreting IFN-γ, and the number of CD4+and CD8+T cells was significantly improved.And HIV-specific antibody, IL-2and IL-4were induced to produce after mmunization with therecombinant virus. Hence, rddVTT-CCMp24could induce robust immune responses.
Then, in order to minimize the development of anti-vector immunity, heterologousprime–boost vaccine strategies have been implemented in mice to enhance the immune responseusing the DNA vaccnie pVL-CCMp24used for priming the immune system and recombinant viralvector vaccine rddVTT-CCMp24as booster. The results of ELISPOT assay had shown that thisprime–boost vaccination could induce to generate more number of antigen-specific IFN-γcompared with either a DNA vector or a live viral vector alone. This graduation thesis provides a foundation for the following empirical study about HIV vaccines.
本研究在本研究室多年来筛选获得的HIV复合多表位基因（MEGNp24）的基础上，在其上游引入CpG基序和CTB基因作为佐剂，构建了一种重组核酸疫苗pVL-CCMp24，将重组DNA质粒转染293T细胞，从RT-PCR和间接免疫荧光检测结果可知，重组质粒编码的目的基因CCMp24在哺乳动物细胞内得到正确转录和表达。肌肉注射途径免疫BALB/c小鼠，分析重组DNA疫苗的免疫原性，由对HIV特异性抗体、Th1型和Th2型细胞因子、T细胞亚型、淋巴细胞增殖等免疫指标的检测结果表明，重组DNA候选疫苗pVL-CCMp24可以诱导机体产生一定程度的细胞和体液免疫应答。
基于多价疫苗的设计理念，本研究首先构建了一种含三个表达盒的痘苗病毒穿梭载体pSTKE，含有三个独立的外源基因表达盒，分别以VTT及其基因缺失突变株作为原始病毒，以增强型绿色荧光蛋白(EGFP)、红色荧光蛋白（RFP）和蓝色荧光蛋白（BFP）作为报告基因，分别对三个表达盒的功能进行验证。通过噬斑筛选，获得两株重组痘苗病毒(rVTT-EGFP、rdVTT-EGFP)，利用PCR、实时荧光定量PCR、Western blot方法对重组病毒以及对外源基因的表达量和遗传稳定性进行分析。结果表明，成功构建了三基因表达盒痘苗病毒穿梭载体，成功筛选出两株重组痘苗病毒，EGFP基因在痘苗病毒内得到高水平表达，三个表达盒之间没有相互影响，而且重组病毒具有良好的遗传稳定性。
将重组核酸疫苗pVL-CCMp24上的目的免疫原基因CCMp24连接到痘苗病毒穿梭载体pSTKE的第一个表达盒内即MCS1，由特异性引物扩增获得的含Loxp基因序列的EGFP基因（EGFP基因两端的Loxp基因方向相同）克隆到pSTKE的第三个表达盒MCS3中，重组质粒pCCMp24-LEL鉴定正确后，转染dVTT感染的BHK-21细胞，通过同源重组、病毒蚀斑筛选获得重组CCMp24和EGFP的E3L及TK基因缺失的痘苗病毒rddVTT-CCMp24G，然后利用Cre/Loxp重组系统将EGFP基因敲除，获得仅表达目的免疫原基因的双基因缺失重组痘苗病毒rddVTT-CCMp24。RT-PCR、间接免疫荧光、Western Blot检测结果表明，CCMp24融合基因在痘苗病毒感染的BHK-21细胞中得到高效转录和表达。
痘苗病毒在长期的实践应用中具有毒副作用，存在安全隐患。本研究在缺失病毒毒力相关基因的痘苗病毒的基础上，通过痘苗病毒穿梭载体pSTKE携带的目的免疫原基因将TK基因缺失，筛选的基因缺失型重组痘苗病毒通过鼻腔感染和颅内注射感染途径感染BALB/c小鼠，通过监测感染小鼠的体征变化、体重变化、病毒感染后的死亡率等方面评价E3L基因和TK基因缺失的重组痘苗病毒在小鼠体内的毒性。监测结果表明，重组病毒rddVTT-CCMp24的毒力较野生型VTT有很大程度的减弱，提高了进一步研究或应用的安全性。说明缺失E3L基因和TK基因可以使痘苗病毒致弱，且外源基因的引入对病毒的毒力没有显著影响。
在了解其毒力的基础上，以BALB/c小鼠为模型开展了重组病毒rddVTT-CCMp24的免疫原性分析。按照既定的免疫程序免疫后，通过对抗体、细胞因子及T细胞亚型等各项指标的检测，结果显示，rddVTT-CCMp24可诱导机体产生分泌IFN-γ的T细胞数量显著增加，CD4+和CD8+T细胞数增加，脾细胞体外接受HIV特异性抗原刺激下增殖能力显著提高。HIV特异性抗体检测结果显示，可诱导较高的HIV特异性抗体水平，且诱导IL-2和IL-4细胞因子的分泌。综合结果可知，重组病毒可激发机体细胞和体液免疫应答。
同时，采用DNA/rddVTT-CCMp24prime-boost免疫策略在小鼠模型上检测免疫效果，结果相对于DNA疫苗或rddVTT-CCMp24疫苗分别单独免疫，可更好的刺激分泌IFN-γ产生T细胞的分化和增殖，IFN-γ ELISPOT检测结果显示，HIV特异性的分泌IFN-γ的记忆性T细胞数量显著高于单独免疫组。表明，prime-boost免疫策略诱导较强的免疫应答和免疫记忆形成，为下一步的实验研究提供了数据支持。
Respecting the currently epidemic of AIDS and its status quo of prevention and therapy, it isimminent to develop a safe and effective AIDS preventative or therapic vaccine. In these years,many scientists had carried out a large number of beneficial trials with various vaccine vectors andmodalities, HIV immunogens, or different routes of vaccination and immunifaction. In thisinvestigation, DNA plasmid and vaccinia virus Tian Tan strain as immunogen delivery systemwere used to launch the trial research of vaccines against HIV.
In this study, we firstly constructed a eukaryotic expression vector of HIV multi-epitope genecontaining unmethylated CpG ODN sequence and cholera toxin B subunit (CTB) and expressionin vitro. CpG-CTB gene sequence (CC) was obtained by PCR using the primers of CTB genecontaining CpG ODN and Linker Sequence designed, and then was connected with pVL. HIVmulti-epitope gene (MEGNp24) from pVAX1-MEGNp24was inserted into the downstream of CCgene to construct pVL-CCMp24. After the recombination plasmid pVL-CCMp24was confirmedby enzyme digestion, it transfected into293T cells with liposome. The expression of CCMp24protein was confirmed by RT-PCR and indirect immunofluorescence. The results showed that theCTB and HIV multi-epitope gene were stably expressed in the293T cells transfected. And thenBALB/c mice were injected with the DNA vaccine by intramuscular injection, to evaluate theimmunogenicity of recombinant DNA vaccine. The immune effects induced by pVL-CCMp24were detected by measuring HIV-specific antibody, Th1type and Th2type cytokines in bloodserum, T cells subtype and lymphocyte proliferation. The results suggested that the recombinantDNA vaccine pVL-CCMp24can induced the production of cellular and humoral immunityresponses in some degree.
Based on the idea of multivalent vaccines and preliminary findings, this study designed andconstructed a vaccinia virus shuttle vector pSTKE with Triple-gene expression cassette. Thevector used vaccinia virus thymidine kinase gene (TK) as screening marker, and contained threeseparate foreign gene expression cassettes. The derived recombinant vaccinia virus can express atleast three different target genes with high efficiency at the whole early and late stages. In addition,knocking out of TK attenuated the virus and improved the safety of clinical application. UsingChinese vaccinia virus Tian Tan strain (VTT) and vaccinia virus Tian Tan strain deleting E3Lgene (dVTT) as original viruses, and enhanced green fluorescent protein (EGFP), red fluorescentprotein (RFP) and blue fluorescent protein (BFP) as reporter gene, we verified the three expressioncassettes. By plaque screening, two recombinant vaccinia viruses (rVTT-EGFP and rdVTT-EGFP)were obtained, and the expression and genetic stability of recombinant virus and foreign genes were analyzed using PCR, real-time quantitative PCR, and Western blot. Results showedsuccessful construction of vaccinia virus shuttle vector with three-gene expression cassette. Twostrains of two recombinant vaccinia viruses were identified, EGFP gene was expressed in thevaccinia virus with a high level, and there was no interaction between the three expressioncassettes, and the two recombinant viruses had good genetic stability.
A HIV immunogen (CCMp24) from pVL-CCMp24was first cloned into MCS1of vacciniavirus shuttle vector pSTKE, and a fragment (LoxP-EGFP-LoxP) containing Loxp sequence andEGFP gene obtained by PCR with the specific primers was inserted into MCS3of shuttle vectorpSTKE, resulting in the recombinant plasmid pCCMp24-LEL. the recombinant plasmid wastransfected into BHK21cells infected with dVTT, and the recombinant virus rddVTT-CCMp24Gwaspurified with plaque isolates expressing green fluorescent protein used as a selection marker, andEGFP gene in the genome of rddVTT-CCMp24Gwas deleted based on Cre/loxp site-specificrecombination, and plaque screeing was performed as described above to generate therecombinant vaccinia virus rddVTT-CCMp24. RT-PCR, indirect immunofluorescence and Westernblot were used to detected the expression of CCMp24in BHK-21cells infected by rddVTT-CCMp24,the results demonsteated that CTB and HIV MEGNp24were expressed in BHK-21cells.
In this research, based on vaccinia virus of virulence related gene deletion, the deletion of TKwas implemented by homologous recombination. The virulence of recombinant virusrddVTT-CCMp24was evaluated in inbred BALB/c mice by the daily measurement of body weightchange after the mice were infected intranasally with rddVTT-CCMp24, ddVTT or VTT.Simultaneously, the neurovirulence of rddVTT-CCMp24was assessed by challenging BALB/c micevia intraperitoneal injection. The neurovirulence in infected mice was determined by measuring50%of the intracranial lethal infectious dose (ICLD50). These results suggest that rddVTT-CCMp24and ddVTT were attenuated due to the deletion of E3L and TK genes, and the introduction of CTBand HIV MEGNp24genes doesn’t alter the infectivity and virulence of the vaccinia virus vectors.
Subsequently, in BALB/c models, the immunogenicity of rddVTT-CCMp24was analyzedthrough measuring HIV-specific antiboady, cytokines in peripheral blood, cellular surface antigenof splenocytes and the number of T cells secreting IFN-γ. We found that the expression of thisimmunogen was sufficient to facilitate the development of substantial number of antigen-specificT cells secreting IFN-γ, and the number of CD4+and CD8+T cells was significantly improved.And HIV-specific antibody, IL-2and IL-4were induced to produce after mmunization with therecombinant virus. Hence, rddVTT-CCMp24could induce robust immune responses.
Then, in order to minimize the development of anti-vector immunity, heterologousprime–boost vaccine strategies have been implemented in mice to enhance the immune responseusing the DNA vaccnie pVL-CCMp24used for priming the immune system and recombinant viralvector vaccine rddVTT-CCMp24as booster. The results of ELISPOT assay had shown that thisprime–boost vaccination could induce to generate more number of antigen-specific IFN-γcompared with either a DNA vector or a live viral vector alone. This graduation thesis provides a foundation for the following empirical study about HIV vaccines.
引文
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